Protease inhibitor peptides

ABSTRACT

Analogues of the Kunitz Protease Inhibitor (KPI) domain of amyloid precursor protein bind to and inhibit activity of serine proteases, including kallikrein, plasmin and coagulation factors such as factors VIIa, IXa, Xa, XIa, and XIIa. Pharmaceutical compositions containing the KPI analogues, along with methods for using such compositions, are useful for ameliorating and treating clinical conditions associated with increased serine protease activity, such as blood loss related to cardiopulmonary bypass surgery. Nucleic acid sequences encoding these analogues and systems for expression of the peptides of the invention are provided.

BACKGROUND OF THE INVENTION

[0001] The plasma, or serine, proteases of the blood contact system areknown to be activated by interaction with negatively charged surfaces.For example, tissue injury during surgery exposes the vascular basementmembrane, causing interaction of the blood with collagen, which isnegatively charged at physiological Ph. This induces a cascade ofproteolytic events, leading to production of plasmin, a fibrinolyticprotease, and consequent blood loss.

[0002] Perioperative blood loss of this type can be particularly severeduring cardiopulmonary bypass (CPB) surgery, in which the patient'sblood flow is diverted to an artificial heart-lung machine. CPB is anessential component of a number of life-saving surgical procedures. Forexample, in the United States, it is estimated that 300,000 patientsevery year undergo coronary artery bypass grafts involving the use ofCPB.

[0003] Although necessary and generally safe, CPB is associated with asignificant rate of morbidity, some of which may be attributed to a“whole body inflammatory response” caused by activation of plasmaprotease systems and blood cells through interactions with theartificial surfaces of the heart-lung machine (Butler et al., Ann.Thorac. Surg. 55:552 (1993); Edmunds et al., J. Card. Surg. 8:404(1993)). For example, during extracorporeal circulation, exposure ofblood to negatively charged surfaces of the artificial bypass circuit,e.g., plastic surfaces in the heart-lung machine, results in directactivation of plasma factor XII.

[0004] Factor XII is a single-chain 80 kDa protein that circulates inplasma as an inactive zymogen. Contact with negatively chargednonendothelial surfaces, like those of the bypass circuit, causessurface-bound factor XII to be autoactivated to the active serineprotease factor XIIa. See Colman, Agents Actions Suppl. 42:125prekallikrein (PK) to active kallikrein, which in turn cleaves more XIIafrom XII in a reciprocal activation reaction that results in a rapidamplification of the contact pathway. Factor XIIa can also activate thefirst component of complement C1, leading to production of theanaphylatoxin C5a through the classical complement pathway.

[0005] The CPB-induced inflammatory response includes changes incapillary permeability and interstitial fluid accumulation. Cleavage ofhigh molecular weight kininogen (HK) by activated kallikrein generatesthe potent vasodilator bradykinin, which is thought to be responsiblefor increasing vascular permeability, resulting in edema, especially inthe lung. The lung is particularly susceptible to damage associated withCPB, with some patients exhibiting what has been called “pump lungsyndrome” following bypass, a condition indistinguishable from adultrespiratory distress. See Johnson et al., J. Thorac. Cardiovasc. Surg.107:1193 (1994).

[0006] Post-CPB pulmonary injury includes tissue damage thought to bemediated by neutrophil sequestration and activation in themicrovasculature of the lung. (Butler et al., supra; Johnson, et al.,supra). Activated factor XII can itself stimulate neutrophilaggregation. Factor XIIa-generated kallikrein, and complement proteinC5a generated by Factor XIIa activation of the complement cascade, bothinduce neutrophil chemotaxis, aggregation and degranulation. See Edmundset al., supra (1993). Activated neutrophils may damage tissue throughrelease of oxygen-derived free-radicals, proteolytic enzymes such aselastase, and metabolites of arachidonic acid. Release of neutrophilproducts in the lung can cause changes in vascular tone, endothelialinjury and loss of vascular integrity.

[0007] Intrinsic inhibition of the contact system occurs throughinhibition of activated XIIa by C1-inhibitor (C1-INH). See Colman,supra. During CPB, this natural inhibitory mechanism is overwhelmed bymassive activation of plasma proteases and consumption of inhibitors. Apotential therapeutic strategy for reducing post-bypass pulmonary injurymediated by neutrophil activation would, therefore, be to block theformation and activity of the neutrophil agonists kallikrein, factorXIIa, and C5a by inhibition of proteolytic activation of the contactsystem.

[0008] Protease inhibitor therapy which partially attenuates the contactsystem is currently employed clinically in CPB. Aprotinin, also known asbasic pancreatic protease inhibitor (BPPI), is a small, basic, 58 aminoacid polypeptide isolated from bovine lung. It is a broad spectrumserine protease inhibitor of the Kunitz type, and was first used duringbypass in an attempt to reduce the inflammatory response to CPB. SeeButler et al., supra. Aprotinin treatment results in a significantreduction in blood loss following bypass, but does not appear tosignificantly reduce neutrophil activation. Additionally, sinceaprotinin is of bovine origin, there is concern that repeatedadministration to patients could lead to the development of an immuneresponse to aprotinin in the patients, precluding its further use.

[0009] The proteases inhibited by aprotinin during CPB appear to includeplasma kallikrein and plasmin. (See, e.g., Scott, et al., Blood 69:1431(1987)). Aprotinin is an inhibitor of plasmin (K_(i) of 0.23 nM), andthe observed reduction in blood loss may be due to inhibition offibrinolysis through the blocking of plasmin action. Although aprotinininhibits plasma kallikrein, (K_(i) of 20 nM), it does not inhibitactivated factor XII, and consequently only partially blocks the contactsystem during CPB.

[0010] Another attractive protease target for use of proteaseinhibitors, such as those of the present invention, is factor XIIa,situated at the very first step of contact activation. By inhibiting theproteolytic activity of factor XIIa, kallikrein production would beprevented, blocking amplification of the contact system, neutrophilactivation and bradykinin release. Inhibition of XIIa would also preventcomplement activation and production of C5a. More complete inhibition ofthe contact system during CPB could, therefore, be achieved through theuse of a better XIIa inhibitor.

[0011] Protein inhibitors of factor XIIa are known. For example, activesite mutants of α₁-antitrypsin that inhibit factor XIIa have been shownto inhibit contact activation in human plasma. See Patston et al., J.Biol. Chem. 265:10786 (1990). The large size and complexity (greaterthan 400 amino acid residues) of these proteins present a significantchallenge for recombinant protein production, since large doses willalmost certainly be required during CPB. For example, although it is apotent inhibitor of both kallikrein and plasmin, nearly 1 gram ofaprotinin must be infused into a patient to inhibit the massiveactivation of the kallikrein-kinin and fibrinolytic systems during CPB.

[0012] The use of smaller, more potent XIIa inhibitors such as the cornand pumpkin trypsin inhibitors (Wen, et al., Protein Exp. & Purif. 4:215(1993); Pedersen, et al., J. Mol. Biol. 236:385 (1994)) could be morecost-effective than the large α₁-antitrypsins, but the infusion of highdoses of these non-mammalian inhibitors could result in immunologicreactions in patients undergoing repeat bypass operations. The idealprotein XIIa inhibitor is, therefore, preferably, small, potent, and ofhuman sequence origin.

[0013] One candidate for an inhibitor of human origin is found incirculating isoforms of the human amyloid β-protein precursor (APPI),also known as protease nexin-2. APPI contains a Kunitz serine proteaseinhibitor domain known as KPI (Kunitz Protease Inhibitor). See Ponte etal., Nature, 331:525 (1988); Tanzi et al., Nature 331:528 (1988);Johnstone et al., Biochem. Biophys. Res. Commun. 163:1248 (1989);Oltersdorf et al., Nature 341:144 (1989). Human KPI shares about 45%amino acid sequence identity with aprotinin. The isolated KPI domain hasbeen prepared by recombinant expression in a variety of systems, and hasbeen shown to be an active serine protease inhibitor. See, for example,Sinha, et al., J. Biol. Chem. 265:8983 (1990). The measured in vitroK_(i) of KPI against plasma kallikrein is 45 nM, compared to 20 nM foraprotinin.

[0014] Aprotinin, KPI, and other Kunitz-type serine protease inhibitorshave been engineered by site-directed mutagenesis to improve inhibitoryactivity or specificity. Thus, substitution of Lys¹⁵ of aprotinin witharginine resulted in an inhibitor with a K_(i) of 0.32 nM toward plasmakallikrein, a 100-fold improvement over natural aprotinin. See PCTapplication No. 89/10374. See also Norris et al., Biol. Chem. HoppeSeyler 371:3742 (1990). Alternatively, substitution of position 15 ofaprotinin with valine or substitution of position 13 of KPI with valineresulted in elastase inhibitors with K_(i)s in the 100 pM range,although neither native aprotinin nor native KPI significantly inhibitselastase. See Wenzel et al., in: Chemistry of Peptides and Proteins,Vol. 3, (Walter de Gruyter, Berlin, New York, 1986); Sinha et al.,supra. Methods for substituting residues 13, 15, 37, and 50 of KPI areshown in general terms in European Patent Application No. 0 393 431, butno specific sequences are disclosed, and no protease inhibition data aregiven.

[0015] Phage display methods have been recently used for preparing andscreening derivatives of Kunitz-type protease inhibitors. See PCTApplication No. 92/15605, which describes specific sequences for 34derivatives of aprotinin, some of which were reportedly active aselastase and cathepsin inhibitors. The amino acid substitutions in thederivatives were distributed throughout almost all positions of theaprotinin molecule.

[0016] Phage display methods have also been used to generate KPIvariants that inhibit factor VIIa and kallikrein. See Dennis et al., J.Biol. Chem. 269:22129 and 269:22137 (1994). The residues that could bevaried in the phage display selection process were limited to positions9-11, 13-17, 32, 36 and 37, and several of those residues were also heldconstant for each selection experiment. One of those variants was saidto have a K_(i) of 1.2 nM for kallikrein, and had substitutions atpositions 9 (Thr→Pro), 13 (Arg→Lys), 15 (Met→Leu), and 37 (Gly→Tyr).None of the inhibitors was tested for the ability to inhibit factorXIIa.

[0017] It is apparent, therefore, that new protease inhibitors that canbind to and inhibit the activity of serine proteases are greatly to bedesired. In particular it is highly desirable to prepare peptides, basedon human peptide sequences, that can inhibit selected serine proteasessuch as kallikrein; chymotrypsins A and B; trypsin; elastase;subtilisin; coagulants and procoagulants, particularly those in activeform, including coagulation factors such as factors VIIa, IXa, Xa, XIa,and XIIa; plasmin; thrombin; proteinase-3; enterokinase; acrosin;cathepsin; urokinase; and tissue plasminogen activator. It is alsohighly desirable to prepare novel protease inhibitors that canameliorate one or more of the undesirable clinical manifestationsassociated with enhanced serine protease activity, for example byreducing pulmonary damage or blood loss during CPB.

SUMMARY OF THE INVENTION

[0018] The present invention relates to peptides that can bind to andpreferably exhibit inhibition of the activity of serine proteases. Thosepeptides can also provide a means of ameliorating, treating orpreventing clinical conditions associated with increased activity ofserine proteases. Particularly, the novel peptides of the presentinvention preferably exhibit a more potent and specific (i.e., greater)inhibitory effect toward serine proteases of interest in comparison toknown serine protease inhibitors. Examples of such proteases include:kallikrein; chymotrypsins A and B; trypsin; elastase; subtilisin;coagulants and procoagulants, particularly those in active form,including coagulation factors such as factors VIIa, IXa, Xa, XIa, andXIIa; plasmin; thrombin; proteinase-3; enterokinase; acrosin; cathepsin;urokinase; and tissue plasminogen activator.

[0019] In achieving the inhibition of serine protease activity, theinvention provides protease inhibitors that can ameliorate one or moreof the undesirable clinical manifestations associated with enhancedserine protease activity, for example, by reducing pulmonary damage orblood loss during CPB.

[0020] The present invention relates to protease inhibitors comprisingthe following amino acid sequences:X¹-Val-Cys-Ser-Glu-Gln-Ala-Glu-X²-Gly-X³-Cys-Arg-Ala-X⁴-X⁵-X⁶-X⁷-Trp-Tyr-Phe-Asp-Val-Thr-Glu-Gly-Lys-Cys-Ala-Pro-Phe-X8-Tyr-Gly-Gly-Cys-X⁹-X¹⁰-X¹¹-X¹²-Asn-Asn-Phe-Asp-Thr-Glu-Glu-Tyr-Cys-Met-Ala-Val-Cys-Gly-Ser-Ala-Ile,

[0021] wherein: X¹ is selected from Glu—Val—Val—Arg—Glu—, Asp, or Glu;X² is selected from Thr, Val, Ile and Ser; X³ is selected from Pro andAla; X⁴ is selected from Arg, Ala, Leu, Gly, or Met; X⁵ is selected fromIle, His, Leu, Lys, Ala, or Phe; X⁶ is selected from Ser, Ile, Pro, Phe,Tyr, Trp, Asn, Leu, His, Lys, or Glu; X⁷ is selected from Arg, His, orAla; X⁸ is selected from Phe, Val, Leu, or Gly; X⁹ is selected from Gly,Ala, Lys, Pro, Arg, Leu, Met, or Tyr; X¹⁰ is selected from Ala, Arg, orGly; X¹¹ is selected from Lys, Ala, or Asn; and X¹² is selected fromSer, Ala, or Arg.

[0022] The invention relates more specifically to protease inhibitorscomprising the following amino acid sequences:X¹-Val-Cys-Ser-Glu-Gln-Ala-Glu-X²-Gly-X³-Cys-Arg-Ala-X⁴-X⁵-X⁶-X⁷-Trp-Tyr-Phe-Asp-Val-Thr-Glu-Gly-Lys-Cys-Ala-Pro-Phe-X⁸-Tyr-Gly-Gly-Cys-X⁹-X¹⁰-X¹¹-X¹²-Asn-Asn-Phe-Asp-Thr-Glu-Glu-Tyr-Cys-Met-Ala-Val-Cys-Gly-Ser-Ala-Ile,

[0023] wherein X¹ is selected from Glu—Val—Val—Arg—Glu—, Asp, or Glu; X²is selected from Thr, Val, Ile and Ser; X³ is selected from Pro and Ala;X⁴ is selected from Arg, Ala, Leu, Gly, or Met; X⁵ is selected from Ile,His, Leu, Lys, Ala, or Phe; X⁶ is selected from Ser, Ile, Pro, Phe, Tyr,Trp, Asn, Leu, His, Lys, or Glu; X⁷ is selected from Arg, His, or Ala;X⁸ is selected from Phe, Val, Leu, or Gly; X⁹ is selected from Gly, Ala,Lys, Pro, Arg, Leu, Met, or Tyr; X¹⁰ is selected from Ala, Arg, or Gly;X¹¹ is selected from Lys, Ala, or Asn; X¹² is selected from Ser, Ala, orArg; provided that when X⁴ is Arg, X⁶ is Ile; when X⁹ is Arg, X⁴ is Alaor Leu; when X⁹ is Tyr, X⁴ is Ala or X⁵ is His; and either X⁵ is notIle; or X⁶ is not Ser; or X⁹ is not Leu, Phe, Met, Tyr, or Asn; or X¹⁰is not Gly; or X¹¹ is not Asn; or X¹² is not Arg.

[0024] Another aspect of this invention provides protease inhibitorswherein at least two amino acid residues selected from the groupconsisting of X⁴, X⁵, X⁶, and X⁷ defined above differ from the residuesfound in the naturally occurring sequence of KPI. Another aspect of thisinvention provides protease inhibitors wherein X¹ is Asp or Glu, X² isThr, X³ is Pro, and X¹² is Ser. Yet another aspect of this inventionprovides protease inhibitors wherein X¹ is Glu, X² is Thr, X³ is Pro, X⁴is Met, X⁵ is Ile, X⁶ is Ser, X⁷ is Arg, x⁸ is Phe, X⁹ is Gly, X¹⁰ isGly, and X¹¹ is Asn. Another aspect of this invention provides proteaseinhibitors wherein X¹ is Asp, X² is Thr, X³ is Pro, X⁴ is Arg, X⁵ isIle, X⁶ is Ile, X⁷ is Arg, x⁸ is Val, X⁹ is Arg, X¹⁰ is Ala, and X¹¹ isLys. Another aspect of this invention provides protease inhibitorswherein X¹ is Glu—Val—Val—Arg—Glu—, X² is Thr, X³ is Pro, X⁴ is Met, X⁵is Ile, X⁶ is Ser, X⁷ is Arg, x⁸ is Phe, X⁹ is Gly, X¹⁰ is Gly, X¹¹ isAsn, and X¹² is Ala. Another aspect of this invention provides proteaseinhibitors wherein X¹ is Glu—Val—Val—Arg—Glu—, X² is Thr, X³ is Pro, X⁴is Met, X⁵ is Ile, X⁶ is Ser, X⁷ is Arg, x⁸ is Phe, X⁹ is Gly, X¹⁰ isGly, X¹¹ is Ala, and X¹² is Arg. Another aspect of this inventionprovides protease inhibitors wherein X¹ is Glu, X² is Thr, X³ is Pro, X⁴is Met, X⁵ is Ile, X⁶ is Ser, X⁷ is Arg, x⁸ is Phe, X⁹ is Gly, X¹⁰ isAla, X¹¹ is Asn, and X¹² is Arg. Another aspect of this inventionprovides protease inhibitors wherein X¹ is Glu—Val—Val—Arg—Glu—, X² isThr, X³ is Pro, X⁴ is Met, X⁵ is Ile, X⁶ is Ser, X⁷ is Arg, x⁸ is Phe,X⁹ is Gly, X¹⁰ is Arg, X¹¹ is Asn, and X¹² is Arg. Another aspect ofthis invention provides protease inhibitors wherein X¹ isGlu—Val—Val—Arg—Glu—, X² is Thr, X³ is Pro, X⁴ is Met, X⁵ is Ile, X⁶ isSer, X⁷ is Arg, x⁸ is Val, Leu, or Gly, X⁹ is Gly, X¹⁰ is Gly, X¹¹ isAsn, and X¹² is Arg. Another aspect of this invention provides proteaseinhibitors wherein X¹ is Glu—Val—Val—Arg—Glu—, X² is Thr, X³ is Pro, X⁴is Met, X⁵ is Ile, X⁶ is Ser, X⁷ is Ala, x⁸ is Phe, X⁹ is Gly, X¹⁰ isGly, X¹¹ is Asn, and X¹² is Arg. Another aspect of this inventionprovides protease inhibitors wherein X¹ is Glu—Val—Val—Arg—Glu—, X² isThr, Val, or Ser, X³ is Pro, X⁴ is Ala or Leu, X⁵ is Ile, X⁶ is Tyr, X⁷His, X⁸ is Phe, X⁹ is Gly, X¹⁰ is Gly, X¹¹ is Ala, and X¹² is Arg.

[0025] Yet another aspect of this invention provides protease inhibitorswherein X² is Thr, and X⁴ is Ala. Another aspect of this inventionprovides protease inhibitors wherein X² is Thr, and X⁴ is Leu. Anotheraspect of this invention provides protease inhibitors wherein X² is Val,and X⁴ is Ala. Another aspect of this invention provides proteaseinhibitors wherein X² is Ser, and X⁴ is Ala. Another aspect of thisinvention provides protease inhibitors wherein X² is Val, and X⁴ is Leu.Another aspect of this invention provides protease inhibitors wherein X²is Ser, and X⁴ is Leu.

[0026] Yet another aspect of this invention provides protease inhibitorswherein X¹ is Glu—Val—Val—Arg—Glu—, X² is Thr, X³ is Pro, X⁴ is Leu, X⁵is Phe, X⁶ is Lys, X⁷ is Arg, X⁸ is Phe, X⁹ is Gly, X¹⁰ is Gly, X¹¹ isAla, and X¹² is Arg. Another aspect of this invention provides proteaseinhibitors wherein X¹ is Glu—Val—Val—Arg—Glu—, X² is Thr, X³ is Pro, X⁴is Leu, X⁵ is Phe, X⁶ is Lys, X⁷ is Arg, X⁸ is Phe, X⁹ is Tyr, X¹⁰ isGly, X¹¹ is Ala, and X¹² is Arg. Another aspect of this inventionprovides protease inhibitors wherein X¹ is Glu—Val—Val—Arg—Glu—, X² isThr, X³ is Pro, X⁴ is Leu, X⁵ is Phe, X⁶ is Lys, X⁷ is Arg, X⁸ is Phe,X⁹ is Leu, X¹⁰ is Gly, X¹¹ is Ala, and X¹² is Arg.

[0027] A further aspect of this invention provides an isolated DNAmolecule comprising a DNA sequence encoding a protease inhibitor of theinvention. Another aspect of this invention provides an isolated DNAmolecule comprising a DNA sequence encoding the protease inhibitor thatfurther comprises an isolated DNA molecule operably linked to aregulatory sequence that controls expression of the coding sequence ofthe protease inhibitor in a host cell. Another aspect of this inventionprovides an isolated DNA molecule comprising a DNA sequence encoding theprotease inhibitor operably linked to a regulatory sequence thatcontrols expression of the coding sequence of the protease inhibitor ina host cell that further comprises a DNA sequence encoding a secretorysignal peptide. That secretory signal peptide may preferably comprisethe signal sequence of yeast alpha-mating factor. Another aspect of thisinvention provides a host cell transformed with any of the DNA moleculesdefined above. Such a host cell may preferably comprise E. coli or ayeast cell. When such a host cell is a yeast cell, the yeast cell maypreferably be Saccharomyces cerevisiae.

[0028] Another aspect of this invention provides a method for producinga protease inhibitor of the present invention, comprising the steps ofculturing a host cell as defined above and isolating and purifying saidprotease inhibitor.

[0029] A further aspect of this invention provides a pharmaceuticalcomposition, comprising a protease inhibitor of the present inventiontogether with a pharmaceutically acceptable sterile vehicle.

[0030] An additional aspect of this invention provides a method oftreatment of a clinical condition associated with increased activity ofone or more serine proteases, comprising administering to a patientsuffering from said clinical condition an effective amount of apharmaceutical composition comprising a protease inhibitor of thepresent invention together with a pharmaceutically acceptable sterilevehicle. That method of treatment may preferably be used to treat theclinical condition of blood loss during surgery.

[0031] Yet another aspect of this invention provides a method forinhibiting the activity of serine proteases of interest in a mammalcomprising administering a therapeutically effective dose of apharmaceutical composition comprising a protease inhibitor of thepresent invention together with a pharmaceutically acceptable sterilevehicle.

[0032] Another aspect of this invention provides a method for inhibitingthe activity of serine proteases of interest in a mammal comprisingadministering a therapeutically effective dose of a pharmaceuticalcomposition comprising a protease inhibitor of the present inventiontogether with a pharmaceutically acceptable sterile vehicle, whereinsaid serine proteases are selected from the group consisting of:kallikrein; chymotrypsins A and B; trypsin; elastase; subtilisin;coagulants and procoagulants, particularly those in active form,including coagulation factors such as factors VIIa, IXa, Xa, XIa, andXIIa; plasmin; thrombin; proteinase-3; enterokinase; acrosin; cathepsin;urokinase; and tissue plasminogen activator.

[0033] A further aspect of this invention relates to protease inhibitorscomprising the following amino acid sequences:X¹-Val-Cys-Ser-Glu-Gln-Ala-Glu-Thr-Gly-Pro-Cys-Arg-Ala-X²-X³-X⁴-Arg-Trp-Tyr-Phe-Asp-Val-Thr-Glu-Gly-Lys-Cys-Ala-Pro-Phe-Phe-Tyr-Gly-Gly-Cys-X⁵-Gly-Asn-Arg-Asn-Asn-Phe-Asp-Thr-Glu-Glu-Tyr-Cys-Met-Ala-Val-Cys-Gly-Ser-Ala-Ile,

[0034] wherein X¹ is selected from Glu—Val—Val—Arg—Glu—, Asp, or Glu; X²is selected from Ala, Leu, Gly, or Met; X³ is selected from Ile, His,Leu, Lys, Ala, or Phe; X⁴ is selected from Ser, Ile, Pro, Phe, Tyr, Trp,Asn, Leu, His, Lys, or Glu; X⁵ is selected from Gly, Ala, Lys, Pro, Arg,Leu, Met, or Tyr; provided that when X⁵ is Arg, X² is Ala or Leu; whenX⁵ is Tyr, X² is Ala or X³ is His; and either X³ is not Ile; or X⁴ isnot Ser; or X⁵ is not Leu, Phe, Met, Tyr, or Asn. Another aspect of thisinvention provides a protease inhibitor as defined above wherein X¹ isGlu, X² is Met, X³ is Ile, X⁴ is Ile, and X⁵ is Gly.

[0035] The invention also relates more specifically to proteaseinhibitors comprising the following amino acid sequences:Glu-Val-Val-Arg-Glu-Val-Cys-Ser-Glu-Gln-Ala-Glu-Thr-Gly-Pro-Cys-Arg-Ala-X¹-X²-X³-Arg-Trp-Tyr-Phe-Asp-Val-Thr-Glu-Gly-Lys-Cys-Ala-Pro-Phe-Phe-Tyr-Gly-Gly-Cys-X⁴-Gly-Asn-Arg-Asn-Asn-Phe-Asp-Thr-Glu-Glu-Tyr-Cys-Met-Ala-Val-Cys-Gly-Ser-Ala-Ile,

[0036] wherein X¹ is selected from Ala, Leu, Gly, or Met; X² is selectedfrom Ile, His, Leu, Lys, Ala, or Phe; X³ is selected from Ser, Ile, Pro,Phe, Tyr, Trp, Asn, Leu, His, Lys, or Glu; X⁴ is selected from Gly, Arg,Leu, Met, or Tyr; provided that when X¹ is Ala, X² is Ile, His, or Leu;when X¹ is Leu, X² is Ile or His; when X¹ is Leu and X² is Ile, X³ isnot Ser; when X¹ is Gly, X² is Ile; when X⁴ is Arg, X¹ is Ala or Leu;when X⁴ is Tyr, X¹ is Ala or X² is His; and either X¹ is not Met, or X²is not Ile, or X³ is not Ser, or X⁴ is not Gly.

[0037] A further aspect of this invention provides a protease inhibitoras defined above wherein X¹ is Met, X³ is Ser, and X⁴ is Gly. Anotheraspect of this invention provides a protease inhibitor wherein X² isselected from His, Ala, Phe, Lys, and Leu. Another aspect of thisinvention provides a protease inhibitor wherein X² is His. Anotheraspect of this invention provides a protease inhibitor wherein X² isAla. Another aspect of this invention provides a protease inhibitorwherein X² is Phe. Another aspect of this invention provides a proteaseinhibitor wherein X² is Lys. Another aspect of this invention provides aprotease inhibitor wherein X² is Leu. Another aspect of this inventionprovides a protease inhibitor wherein X¹ is Met, X² is Ile, and X⁴ isGly.

[0038] Yet another aspect of this invention provides a proteaseinhibitor wherein X³ is Ile. Another aspect of this invention provides aprotease inhibitor wherein X³ is Pro. Another aspect of this inventionprovides a protease inhibitor wherein X³ is Phe. Another aspect of thisinvention provides a protease inhibitor wherein X³ is Tyr. Anotheraspect of this invention provides a protease inhibitor wherein X³ isTrp. Another aspect of this invention provides a protease inhibitorwherein X³ is Asn. Another aspect of this invention provides a proteaseinhibitor wherein X³ is Leu.

[0039] An additional aspect of this invention provides a proteaseinhibitor wherein X³ is Lys. Another aspect of this invention provides aprotease inhibitor wherein X³ is His. Another aspect of this inventionprovides a protease inhibitor wherein X³ is Glu. Another aspect of thisinvention provides a protease inhibitor wherein X¹ is Ala. Anotheraspect of this invention provides a protease inhibitor wherein X² isIle. Another aspect of this invention provides a protease inhibitorwherein X³ is Phe, and X⁴ is Gly. Another aspect of this inventionprovides a protease inhibitor wherein X³ is Tyr, and X⁴ is Gly. Anotheraspect of this invention provides a protease inhibitor wherein X³ isTrp, and X⁴ is Gly.

[0040] Yet another other aspect of this invention provides a proteaseinhibitor wherein X³ is Ser or Phe, and X⁴ is Arg or Tyr. Another aspectof this invention provides a protease inhibitor wherein X² is His orLeu, X³ is Phe, and X⁴ is Gly. Another aspect of this invention providesa protease inhibitor wherein X¹ is Leu. Another aspect of this inventionprovides a protease inhibitor wherein X² is His, X³ is Asn or Phe, andX⁴ is Gly. Another aspect of this invention provides a proteaseinhibitor wherein X² is Ile, X³ is Pro, and X⁴ is Gly. Another aspect ofthis invention provides a protease inhibitor wherein X¹ is Gly, X² isIle, X³ is Tyr, and X⁴ is Gly. Another aspect of this invention providesa protease inhibitor wherein X¹ is Met, X² is His, X³ is Ser, and X⁴ isTyr.

[0041] Additionally, another aspect of this invention relates toprotease inhibitors comprising the following amino acid sequences:X¹-Val-Cys-Ser-Glu-Gln-Ala-Glu-X²-Gly-Pro-Cys-Arg-Ala-X³-X⁴-X⁵-X⁶-Arg-Trp-Tyr-Phe-Asp-Val-Thr-Glu-Gly-Lys-Cys-Ala-Pro-Phe-Phe-Tyr-Gly-Gly-Cys-X⁷-Gly-Asn-Arg-Asn-Asn-Phe-Asp-Thr-Glu-Glu-Tyr-Cys-Met-Ala-Val-Cys-Gly-Ser-Ala-Ile,

[0042] wherein X¹ is selected from Glu—Val—Val—Arg—Glu—, Asp, or Glu; X²is selected from Thr, Val, Ile and Ser; X³ is selected from Arg, Ala,Leu, Gly, or Met; X⁴ is selected from Ile, His, Leu, Lys, Ala, or Phe;X⁵ is selected from Ser, Ile, Pro, Phe, Tyr, Trp, Asn, Leu, His, Lys, orGlu; X⁶ is selected from Arg, His, or Ala; and X⁷ is selected from Gly,Ala, Lys, Pro, Arg, Leu, Met, or Tyr.

[0043] Another aspect of this invention provides a protease inhibitor asdefined above wherein at least two amino acid residues selected from thegroup consisting of X³, X⁴, X^(5,) and X⁶ differ from the residues foundin the naturally occurring sequence of KPI. Another aspect of thisinvention provides a protease inhibitor wherein X¹ isGlu—Val—Val—Arg—Glu—, X² is Thr, Val, or Ser, X³ is Ala or Leu, X⁴ isIle, X⁵ is Tyr, X⁶ is His and X⁷ is Gly. Another aspect of thisinvention provides a protease inhibitor wherein X² is Thr, and X³ isAla. Another aspect of this invention provides a protease inhibitorwherein X² is Thr, and X³ is Leu. Another aspect of this inventionprovides a protease inhibitor wherein X² is Val, and X³ is Ala. Anotheraspect of this invention provides a protease inhibitor wherein X² isSer, and X³ is Ala. Another aspect of this invention provides a proteaseinhibitor wherein X² is Val, and X³ is Leu. Another aspect of thisinvention provides a protease inhibitor wherein X² is Ser, and X³ isLeu. Another aspect of this invention provides a protease inhibitorwherein X¹ is Glu—Val—Val—Arg—Glu—, X² is Thr, X³ is Leu, X⁴ is Phe, X⁵is Lys, X⁶ is Arg and X⁷ is Gly. Another aspect of this inventionprovides a protease inhibitor wherein X¹ is Glu—Val—Val—Arg—Glu—, X² isThr, X³ is Leu, X⁴ is Phe, X⁵ is Lys, X⁶ is Arg and X⁷ is Tyr. Anotheraspect of this invention provides a protease inhibitor wherein X¹ isGlu—Val—Val—Arg—Glu—, X² is Thr, X³ is Leu, X⁴ is Phe, X⁵ is Lys, X⁶ isArg and X⁷ is Leu.

[0044] Other objects, features and advantages of the present inventionwill become apparent from the following detailed description. It shouldbe understood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0045]FIG. 1 shows the strategy for the construction of plasmidpTW10:KPI.

[0046]FIG. 2 shows the sequence of the synthetic gene for KPI (1→57)fused to the bacterial phoA secretory signal sequence.

[0047]FIG. 3 shows the strategy for construction of plasmid pKPI-61.

[0048]FIG. 4 shows the 192 bp XbaI-HindIII synthetic gene fragmentencoding KPI (1→57) and four amino acids from yeast alpha-mating factor.

[0049]FIG. 5 shows the synthetic 201 bp XbaI-HindIII fragment encodingKPI(−4→57) in PKPI-61.

[0050]FIG. 6 shows the strategy for the construction of plasmid pTW113.

[0051]FIG. 7 shows plasmid PTW113, encoding the 445 bp synthetic genefor yeast alpha-factor-KPI(−4→57) fusion.

[0052]FIG. 8 shows the amino acid sequence for KPI (−4→57).

[0053]FIG. 9 shows the strategy for constructing plasmid pTW6165.

[0054]FIG. 10 shows plasmid, PTW6165, encoding the 445 bp synthetic genefor alpha-factor-KPI(−4→57; M15A, S17W) fusion.

[0055]FIG. 11 shows the sequences of the annealed oligonucleotide pairsused to construct plasmids PTW6165, pTW6166, pTW6175, pBG028, pTW6183,pTW6184, pTW6185, pTW6173, and pTW6174.

[0056]FIG. 12 shows the sequence of plasmid PTW6166 encoding the fusionof yeast alpha-factor and KPI(−4→57; M15A, S17Y).

[0057]FIG. 13 shows the sequence of plasmid PTW6175 encoding the fusionof yeast alpha-factor and KPI(−4→57; M15L, S17F).

[0058]FIG. 14 shows the sequence of plasmid PBG028 encoding the fusionof yeast alpha-factor and KPI(−4→57; M15L, S17Y).

[0059]FIG. 15 shows the sequence of plasmid PTW6183 encoding the fusionof yeast alpha-factor and KPI(−4→57; I16H, S17F).

[0060]FIG. 16 shows the sequence of plasmid PTW6184 encoding the fusionof yeast alpha-factor and KPI(−4→57; I16H, S17Y).

[0061]FIG. 17 shows the sequence of plasmid PTW6185 encoding the fusionof yeast alpha-factor and KPI(−4→57; I16H, S17W).

[0062]FIG. 18 shows the sequence of plasmid PTW6173 encoding the fusionof yeast alpha-factor and KPI(−4→57; M15A, I16H).

[0063]FIG. 19 shows the sequence of plasmid PTW6174 encoding the fusionof yeast alpha-factor and KPI(−4→57; M15L, I16H).

[0064]FIG. 20 shows the amino acid sequence of KPI (−4→57; M15A, S17W).

[0065]FIG. 21 shows the amino acid sequence of KPI (−4→57; M15A, S17Y).

[0066]FIG. 22 shows the amino acid sequence of KPI (−4→57; M15L, S17F).

[0067]FIG. 23 shows the amino acid sequence of KPI (−4→57; M15L, S17Y).

[0068]FIG. 24 shows the amino acid sequence of KPI (−4→57; I16H, S17F).

[0069]FIG. 25 shows the amino acid sequence of KPI (−4→57; I16H, S17Y).

[0070]FIG. 26 shows the amino acid sequence of KPI (−4→57; I16H, S17W).

[0071]FIG. 27 shows the amino acid sequence of KPI (−4→57; M15A, S17F).

[0072]FIG. 28 shows the amino acid sequence of KPI (−4→57; M15A, I16H).

[0073]FIG. 29 shows the amino acid sequence of KPI (−4→57; M15L, I16H).

[0074]FIG. 30 shows the construction of plasmid pSP26:Amp:F1.

[0075]FIG. 31 shows the construction of plasmid pgIII.

[0076]FIG. 32 shows the construction of plasmid pPhoA:KPI:gIII.

[0077]FIG. 33 shows the construction of plasmid pLG1.

[0078]FIG. 34 shows the construction of plasmid pAL51.

[0079]FIG. 35 shows the construction of plasmid pAL53.

[0080]FIG. 36 shows the construction of plasmidPSP26:Amp:F1:PhoA:KPI:gIII.

[0081]FIG. 37 shows the construction of plasmid pDW1 #14.

[0082]FIG. 38 shows the coding region for the fusion of phoA-KPI(1→55)-geneIII.

[0083]FIG. 39 shows the construction of plasmid PDW1 14-2.

[0084]FIG. 40 shows the construction of KPI Library 16-19.

[0085]FIG. 41 shows the expression unit encoded by the members of KPILibrary 16-19.

[0086]FIG. 42 shows the phoA-KPI(1→55)-geneIII region encoded by themost frequently occurring randomized KPI region.

[0087]FIG. 43 shows the construction of pDD185 KPI (−4→57; M15A, S17F).

[0088]FIG. 44 shows the sequence of alpha-factor fused to KPI (−4→57;M15A, S17F).

[0089]FIG. 45 shows the inhibition constants (K_(i)s) determined forpurified KPI variants against the selected serine proteases kallikrein,factor Xa, and factor XIIa.

[0090]FIG. 46 shows the inhibition constants (K_(i)s) determined for KPIvariants against kallikrein, plasmin, and factors Xa, XIa, and XIIa.

[0091]FIG. 47 shows the post-surgical blood loss in pigs in the presence(KPI) and absence (NS) of KPI 185-1 (M15A, S17F).

[0092]FIG. 48 shows the post-surgical hemoglobin loss in pigs in thepresence (KPI) and absence (NS) of KPI 185-1 (M15A, S17F).

[0093]FIG. 49 shows the oxygen tension in the presence and absence ofKPI, before CPB, immediately after CPB, and at 60 and 180 minutes afterthe end of CPB.

[0094]FIG. 50 summarizes the results shown in FIGS. 47-49.

DETAILED DESCRIPTION

[0095] The present invention provides peptides that can bind to andpreferably inhibit the activity of serine proteases. These inhibitorypeptides can also provide a means of ameliorating, treating orpreventing clinical conditions associated with increased activity ofserine proteases. The novel peptides of the present invention preferablyexhibit a more potent and specific (i.e., greater) inhibitory effecttoward serine proteases of interest than known serine proteaseinhibitors. Examples of such proteases include: kallikrein;chymotrypsins A and B; trypsin; elastase; subtilisin; coagulants andprocoagulants, particularly those in active form, including coagulationfactors such as factors VIIa, IXa, Xa, XIa, and XIIa; plasmin; thrombin;proteinase-3; enterokinase; acrosin; cathepsin; urokinase; and tissueplasminogen activator.

[0096] Peptides of the present invention may be used to reduce thetissue damage caused by activation of the proteases of the contactpathway of the blood during surgical procedures such as cardiopulmonarybypass (CPB). Inhibition of contact pathway proteases reduces the “wholebody inflammatory response” that can accompany contact pathwayactivation, and that can lead to tissue damage, and possibly death. Thepeptides of the present invention may also be used in conjunction withsurgical procedures to reduce activated serine protease-associatedperioperative and postoperative blood loss. For instance, perioperativeblood loss of this type may be particularly severe during CPB surgery.Pharmaceutical compositions comprising the peptides of the presentinvention may be used in conjunction with surgery such as CPB;administration of such compositions may occur preoperatively,perioperatively or postoperatively. Examples of other clinicalconditions associated with increased serine protease activity for whichthe peptides of the present invention may be used include: CPB-inducedinflammatory response; post-CPB pulmonary injury; pancreatitis;allergy-induced protease release; deep vein thrombosis;thrombocytopenia; rheumatoid arthritis; adult respiratory distresssyndrome; chronic inflammatory bowel disease; psoriasis;hyperfibrinolytic hemorrhage; organ preservation; wound healing; andmyocardial infarction. Other examples of preferable uses of the peptidesof the present invention are described in U.S. Pat. No. 5,187,153.

[0097] The invention is based upon the novel substitution of amino acidresidues in the peptide corresponding to the naturally occurring KPIprotease inhibitor domain of human amyloid β-amyloid precursor protein(APPI). These substitutions produce peptides that can bind to serineproteases and preferably exhibit an inhibition of the activity of serineproteases. The peptides also preferably exhibit a more potent andspecific serine protease inhibition than known serine proteaseinhibitors. In accordance with the invention, peptides are provided thatmay exhibit a more potent and specific inhibition of one or more serineproteases of interest, e.g., kallikrein, plasmin and factors Xa, XIa,XIIa, and XIIa.

[0098] The present invention also includes pharmaceutical compositionscomprising an effective amount of at least one of the peptides of theinvention, in combination with a pharmaceutically acceptable sterilevehicle, as described in REMINGTON'S PHARMACEUTICAL SCIENCES: DRUGRECEPTORS AND RECEPTOR THEORY, (18th ed.), Mack Publishing Co., Easton,Pa. (1990).

[0099] A. Selection of Sequences of KPI Variants

[0100] The sequence of KPI is shown in Table 1. Table 2 shows acomparison of this sequence with that of aprotinin, with which it sharesabout 45% sequence identity. The numbering convention for KPI shown inTable 1 and used hereinafter designates the first glutamic acid residueof KPI as residue 1. This corresponds to residue number 3 using thestandard numbering convention for aprotinin.

[0101] The crystal structure for KPI complexed with trypsin has beendetermined. See Perona et al., J. Mol. Biol. 230:919 (1993). Thethree-dimensional structure reveals two binding loops within KPI thatcontact the protease. The first loop extends from residue Thr⁹ to Ile¹⁶,and the second loop extends from residue Phe³² to Gly³⁷. The twoprotease binding loops are joined through the disulfide bridge extendingfrom Cys¹² to Cys³⁶. KPI contains two other disulfide bridges, betweenCys³ and Cys⁵³, and between Cys²⁸ to Cys⁴⁹.

[0102] This structure was used as a guide to inform our strategy formaking the amino acid residue substitutions that will be most likely toaffect the protease inhibitory properties of KPI. Our examination of thestructure indicated that certain amino acid residues, including residues9, 11, 13-18, 32, and 37-40, appear to be of particular significance indetermining the protease binding properties of the KPI peptide. In apreferred embodiment of the invention two or more of those KPI peptideresidues are substituted; such substitutions preferably occurring amongresidues 9, 11, 13-18, 32, and 37-40. In particular, we found that thosesubstituted peptides, including peptides comprising substitutions of atleast two of the four residues at positions 15-18, may exhibit morepotent and specific serine protease inhibition toward selected serineproteases of interest than exhibited by the natural KPI peptide domain.Such substituted peptides may further comprise one or more additionalsubstitutions at residues 9, 11, 13, 14, 32 and 37-40; in particular,such peptides may further comprise a substitution at positions 9 or 37.In particular, the peptides of the present invention preferably exhibita greater potency and specificity for inhibiting one or more serineproteases of interest (e.g., kallikrein, plasmin and factors VIIa, IXa,Xa, XIa, and XIIa) than the potency and specificity exhibited by nativeKPI or other known serine protease inhibitors. That greater potency andspecificity may be manifested by the peptides of the present inventionby exhibiting binding constants for serine proteases of interest thatare less than the binding constants exhibited by native KPI, or otherknown serine protease inhibitors, for such proteases.

[0103] By way of example, and as set forth in greater detail below, theserine protease inhibitory properties of peptides of the presentinvention were measured for the serine proteases of interest—kallikrein,plasmin and factors Xa, XIa, and XIIa. Methodologies for measuring theinhibitory properties of the KPI variants of the present invention areknown to those skilled in the art, e.g., by determining the inhibitionconstants of the variants toward serine proteases of interest, asdescribed in Example 4, infra. Such studies measure the ability of thenovel peptides of the present invention to bind to one or more serineproteases of interest and to preferably exhibit a greater potency andspecificity for inhibiting one or more serine protease of interest thanknown serine protease inhibitors such as native KPI.

[0104] The ability of the peptides of the present invention to bind oneor more serine proteases of interest, particularly the ability of thepeptides to exhibit such greater potency and specificity toward serineproteases of interest, manifest the clinical and therapeuticapplications of such peptides. The clinical and therapeutic efficacy ofthe peptides of the present invention can be assayed by in vitro and invivo methodologies known to those skilled in the art, e.g., as describedin Example 5, infra. TABLE 1 SEQUENCE OF KPI:   1                 10                  20                  30 V R E VC S E Q A E T G P C R A M I S R W Y F D V T E G K C A P                  40                 50 F F Y G G C G G N R N N F D T EE Y C M A V C G S A I

[0105] TABLE 2 COMPARISON OF KPI AND APROTININ SEQUENCES:   1            10        20  30        40        50 KPI:VREVCSEQAETGPCRAMISRWYFDVTEGKCAPFFYGGCGGNRNNFDTEEYCMAVCGSAI    | |   |||| | | |       | |  | ||||   ||||   | ||  || | BPTI:RPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTCGGA1       10        20        30        40        50

[0106] B. Methods of Producing KPI Variants

[0107] The peptides of the present invention can be created by synthetictechniques or recombinant techniques which employ genomic or cDNAcloning methods.

[0108] 1. Production by chemical synthesis

[0109] Peptides of the present invention can be routinely synthesizedusing solid phase or solution phase peptide synthesis. Methods ofpreparing relatively short peptides such as KPI by chemical synthesisare well known in the art. KPI variants could, for example be producedby solid-phase peptide synthesis techniques using commercially availableequipment and reagents such as those available from Milligen (Bedford,Mass.) or Applied Biosystems-Perkin Elmer (Foster City, Calif.).Alternatively, segments of KPI variants could be prepared by solid-phasesynthesis and linked together using segment condensation methods such asthose described by Dawson et al., Science 266:776 (1994). Duringchemical synthesis of the KPI variants, substitution of any amino acidis achieved simply by replacement of the residue that is to besubstituted with a different amino acid monomer.

[0110] 2. Production by recombinant DNA technology

[0111] (a) Preparation of genes encoding KPI variants

[0112] In a preferred embodiment of the invention, KPI variants areproduced by recombinant DNA technology. This requires the preparation ofgenes encoding each KPI variant that is to be made. Suitable genes canbe constructed by oligonucleotide synthesis using commercially availableequipment, such as that provided by Milligen and Applied Biosystems,supra. The genes can be prepared by synthesizing the entire coding andnon-coding strands, followed by annealing the two strands.Alternatively, the genes can be prepared by ligation of smallersynthetic oligonucleotides by methods well known in the art. Genesencoding KPI variants are produced by varying the nucleotides introducedat any step of the synthesis to change the amino acid sequence encodedby the gene.

[0113] Preferably, however, KPI variants are made by site-directedmutagenesis of a gene encoding KPI. Methods of site-directed mutagenesisare well known in the art. See, for example, Ausubel et al., (eds.)CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Wiley Interscience, 1987);PROTEIN ENGINEERING (Oxender & Fox eds., A. Liss, Inc. 1987). Thesemethods require the availability of a gene encoding KPI or a variantthereof, which can then be mutagenized by known methods to produce thedesired KPI variants. In addition, linker-scanning and polymerase chainreaction (“PCR”) mediated techniques can be used for purposes ofmutagenesis. See PCR TECHNOLOGY (Erlich ed., Stockton Press 1989);CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, vols. 1 & 2, loc. cit.

[0114] A gene encoding KPI can be obtained by cloning the naturallyoccurring gene, as described for example in U.S. Pat. Nos. 5,223,482 and5,187,153, which are hereby incorporated by reference in theirentireties. In particular, see columns 6-9 of U.S. Pat. No. 5,187,153.See also PCT Application No. 93/09233. In a preferred embodiment of theinvention a synthetic gene encoding KPI is produced by chemicalsynthesis, as described above. The gene may encode the 57-amino acid KPIdomain shown in Table 1, or it may also encode additional N-terminalamino acids from the APPI protein sequence, such as the four amino acidsequence (Glu—Val—Val—Arg, designated residues −4 to −1) immediatelypreceding the KPI domain in APPI.

[0115] Production of the gene by synthesis allows the codon usage of theKPI gene to be altered to introduce convenient restriction endonucleaserecognition sites, without altering the sequence of the encoded peptide.In a preferred embodiment of the invention, the synthetic KPI genecontains restriction endonuclease recognition sites that facilitateexcision of DNA cassettes from the KPI gene. These cassettes can bereplaced with small synthetic oligonucleotides encoding the desiredchanges in the KPI peptide sequence. See Ausubel, supra.

[0116] This method also allows the production of genes encoding KPI as afusion peptide with one or more additional peptide or protein sequences.The DNA encoding these additional sequences is arranged in-frame withthe sequence encoding KPI such that, upon translation of the gene, afusion protein of KPI and the additional peptide or protein sequence isproduced. Methods of making such fusion proteins are well known in theart. Examples of additional peptide sequences that can be encoded in thegenes are secretory signal peptide sequences, such as bacterial leadersequences, for example ompA and phoA, that direct secretion of proteinsto the bacterial periplasmic space. In a preferred embodiment of theinvention, the additional peptide sequence is a yeast secretory signalsequence, such as α-mating factor, that directs secretion of the peptidewhen produced in yeast.

[0117] Additional genetic regulatory sequences can also be introducedinto the synthetic gene that are operably linked to the coding sequenceof the gene, thereby allowing synthesis of the protein encoded by thegene when the gene is introduced into a host cell. Examples ofregulatory genetic sequences that can be introduced are: promoter andenhancer sequences and transcriptional and translational controlsequences. Other regulatory sequences are well known in the art. SeeAusubel et al., supra, and Sambrook et al., supra.

[0118] Sequences encoding other fusion proteins and genetic elements arewell known to those of skill in the art. In a preferred embodiment ofthe invention, the KPI sequence is prepared by ligating togethersynthetic oligonucleotides to produce a gene encoding an in-frame fusionprotein of yeast α-mating factor with either KPI (1→57) or KPI (−4→57).

[0119] The gene constructs prepared as described above are convenientlymanipulated in host cells using methods of manipulating recombinant DNAtechniques that are well known in the art. See, for example Sambrook etal., MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1989), andAusubel, supra. In a preferred embodiment of the invention the host cellused for manipulating the KPI constructs is E. coli. For example, theconstruct can be ligated into a cloning vector and propagated in E. coliby methods that are well known in the art. Suitable cloning vectors aredescribed in Sambrook, supra, or are commercially available fromsuppliers such as Promega (Madison, Wis.), Stratagene (San Diego,Calif.) and Life Technologies (Gaithersburg, Md.).

[0120] Once a gene construct encoding KPI has been obtained, genesencoding KPI variants are obtained by manipulating the coding sequenceof the construct by standard methods of site-directed mutagenesis, suchas excision and replacement of small DNA cassettes, as described supra.See Ausubel, supra, and Sinha et al., supra. See also U.S. Pat. No.5,373,090, which is herein incorporated by reference in its entirety.See particularly, columns 4-12 of U.S. Pat. No. 5,272,090. These genesare then used to produce the KPI variant peptides as described below.

[0121] Alternatively, KPI variants can be produced using phage displaymethods. See, for example, Dennis et al. supra, which is herebyincorporated by reference in its entirety. See also U.S. Pat. Nos.5,223,409 and 5,403,484, which are hereby also incorporated by referencein their entireties. In these methods, libraries of genes encodingvariants of KPI are fused in-frame to genes encoding surface proteins offilamentous phage, and the resulting peptides are expressed (displayed)on the surface of the phage. The phage are then screened for the abilityto bind, under appropriate conditions, to serine proteases of interestimmobilized on a solid support. Large libraries of phage can be used,allowing simultaneous screening of the binding properties of a largenumber of KPI variants. Phage that have desirable binding properties areisolated and the sequences of the genes encoding the corresponding KPIvariants is determined. These genes are then used to produce the KPIvariant peptides as described below.

[0122] (b) Expression of KPI variant peptides

[0123] Once genes encoding KPI variants have been prepared, they areinserted into an expression vector and used to produce the recombinantpeptide. Suitable expression vectors and corresponding methods ofexpressing recombinant proteins and peptides are well known in the art.Methods of expressing KPI peptides are described in U.S. Pat. No.5,187,153, columns 9-11, U.S. Pat. No. 5,223,482, columns 9-11, and PCTapplication 93/09233, pp. 49-67. See also Ausubel et al., supra, andSambrook et al., supra. The gene can be expressed in any number ofdifferent recombinant DNA expression systems to generate large amountsof the KPI variant, which can then be purified and tested for itsability to bind to and inhibit serine proteases of interest.

[0124] Examples of expression systems known to the skilled practitionerin the art include bacteria such as E. coli, yeast such as Saccharomycescerevisiae and Pichia pastoris, baculovirus, and mammalian expressionsystems such as in Cos or CHO cells. In a preferred embodiment, KPIvariants are expressed in S. cerevisiae. In another preferred embodimentthe KPI variants are cloned into expression vectors to produce achimeric gene encoding a fusion protein of the KPI variant with yeastα-mating factor. The mating factor acts as a signal sequence to directsecretion of the fusion protein from the yeast cell, and is then cleavedfrom the fusion protein by a membrane-bound protease during thesecretion process. The expression vector is transformed into S.cerevisiae, the transformed yeast cells are cultured by standardmethods, and the KPI variant is purified from the yeast growth medium.

[0125] Recombinant bacterial cells expressing the peptides of thepresent invention, for example, E. coli, are grown in any of a number ofsuitable media, for example LB, and the expression of the recombinantantigen induced by adding IPTG to the media or switching incubation to ahigher temperature. After culturing the bacteria for a further period ofbetween 2 and 24 hours, the cells are collected by centrifugation andwashed to remove residual media. The bacterial cells are then lysed, forexample, by disruption in a cell homogenizer and centrifuged to separatedense inclusion bodies and cell membranes from the soluble cellcomponents. This centrifugation can be performed under conditionswhereby dense inclusion bodies are selectively enriched by incorporationof sugars such as sucrose into the buffer and centrifugation at aselective speed. If the recombinant peptide is expressed in inclusionbodies, as is the case in many instances, these can be washed in any ofseveral solutions to assist in the removal of any contaminating hostproteins, then solubilized in solutions containing high concentrationsof urea (e.g., 8M) or chaotropic agents such as guanidine hydrochloridein the presence of reducing agents such as β-mercaptoethanol or DTT(dithiothreitol).

[0126] At this stage it may be advantageous to incubate the peptides ofthe present invention for several hours under conditions suitable forthe peptides to undergo a refolding process into a conformation whichmore closely resembles that of native KPI. Such conditions generallyinclude low protein concentrations less than 500 μg/ml, low levels ofreducing agent, concentrations of urea less than 2M and often thepresence of reagents such as a mixture of reduced and oxidizedglutathione which facilitate the interchange of disulphide bonds withinthe protein molecule. The refolding process can be monitored, forexample, by SDS-PAGE or with antibodies which are specific for thenative molecule (which can be obtained from animals vaccinated with thenative molecule isolated from parasites). Following refolding, thepeptide can then be purified further and separated from the refoldingmixture by chromatography on any of several supports including ionexchange resins, gel permeation resins or on a variety of affinitycolumns.

[0127] Purification of KPI variants can be achieved by standard methodsof protein purification, e.g., using various chromatographic methodsincluding high performance liquid chromatography and adsorptionchromatography. The purity and the quality of the peptides can beconfirmed by amino acid analyses, molecular weight determination,sequence determination and mass spectrometry. See, for example, PROTEINPURIFICATION METHODS—A PRACTICAL APPROACH, Harris et al., eds. (IRLPress, Oxford, 1989). In a preferred embodiment, the yeast cells areremoved from the growth medium by filtration or centrifugation, and theKPI variant is purified by affinity chromatography on a column oftrypsin-agarose, followed by reversed-phase HPLC.

[0128] C. Measurement of Protease Inhibitory Properties of KPI Variants

[0129] Once KPI variants have been purified, they are tested for theirability to bind to and inhibit serine proteases of interest in vitro.The peptides of the present invention preferably exhibit a more potentand specific inhibition of serine proteases of interest than knownserine protease inhibitors, such as the natural KPI peptide domain. Suchbinding and inhibition can be assayed for by determining the inhibitionconstants for the peptides of the present invention toward serineproteases of interest and comparing those constants with constantsdetermined for known serine protease inhibitors, e.g., the native KPIdomain, toward those proteases. Methods for determining inhibitionconstants of protease inhibitors are well known in the art. See Fersht,ENZYME STRUCTURE AND MECHANISM, 2nd ed., W. H. Freeman and Co., NewYork, (1985).

[0130] In a preferred embodiment the inhibition experiments are carriedout using a chromogenic synthetic protease substrate, as described, forexample, in Bender et al., J. Amer. Chem. Soc. 88:5890 (1966).Measurements taken by this method can be used to calculate inhibitionconstants (K_(i) values) of the peptides of the present invention towardserine proteases of interest. See Bieth in BAYER-SYMPOSIUM V “PROTEINASEINHIBITORS”, Fritz et al., eds., pp. 463-69, Springer-Verlag, Berlin,Heidelberg, New York, (1974). KPI variants that exhibit potent andspecific inhibition of one or more serine proteases of interest maysubsequently be tested in vivo. In vitro testing, however, is not aprerequisite for in vivo studies of the peptides of the presentinvention.

[0131] D. Testing of KPI Variants in vivo

[0132] The peptides of the present invention may be tested, alone or incombination, for their therapeutic efficacy by various in vivomethodologies known to those skilled in the art, e.g., the ability ofKPI variants to reduce postoperative bleeding can be tested in standardanimal models. For example, cardiopulmonary bypass surgery can becarried out on animals such as pigs in the presence of KPI variants, orin control animals where the KPI variant is not used. The use of pigs asa model for studying the clinical effects associated with CPB haspreviously been described. See Redmond et al., Ann. Thorac. Surg. 56:474(1993).

[0133] The KPI variant is supplied to the animals in a pharmaceuticalsterile vehicle by methods known in the art, for example by continuousintravenous infusion. Chest tubes can be used to collect shed blood fora defined period of time. The shed blood, together with the residualintrathoracic blood found after sacrifice of the animal can be used tocalculate hemoglobin (Hgb) loss. The postoperative blood and Hgb loss isthen compared between the test and control animals to determine theeffect of the KPI variants.

[0134] E. Therapeutic Use of KPI Variants

[0135] KPI variants of the present invention found to exhibittherapeutic efficacy (e.g., reduction of blood loss following surgery inanimal models) may preferably be used and administered, alone or incombination or as a fusion protein, in a manner analogous to thatcurrently used for aprotinin or other known serine protease inhibitors.See Butler et al., supra. Peptides of the present invention generallymay be administered in the manner that natural peptides areadministered. A therapeutically effective dose of the peptides of thepresent invention preferably affects the activity of the serineproteases of interest such that the clinical condition may be treated,ameliorated or prevented. Therapeutically effective dosages of thepeptides of the present invention can be determined by those skilled inthe art, e.g., through in vivo or in vitro models. Generally, thepeptides of the present invention may be administered in total amountsof approximately 0.01 to approximately 500, specifically 0.1 to 100mg/kg body weight, if desired in the form of one or moreadministrations, to achieve therapeutic effect. It may, however, benecessary to deviate from such administration amounts, in particulardepending on the nature and body weight of the individual to be treated,the nature of the medical condition to be treated, the type ofpreparation and the administration of the peptide, and the time intervalover which such administration occurs. Thus, it may in some cases besufficient to use less than the above amount of the peptides of thepresent invention, while in other cases the above amount is preferablyexceeded. The optimal dose required in each case and the type ofadministration of the peptides of the present invention can bedetermined by one skilled in the art in view of the circumstancessurrounding such administration. Such peptides can be administered byintravenous injections, in situ injections, local applications,inhalation, oral administration using coated polymers, dermal patches orother appropriate means. Compositions comprising peptides of the presentinvention are advantageously administered in the form of injectablecompositions. Such peptides may be preferably administered to patientsvia continuous intravenous infusion, but can also be administered bysingle or multiple injections. A typical composition for such purposecomprises a pharmaceutically acceptable carrier. Pharmaceuticallyacceptable carriers include aqueous solutions, non-toxic excipients,including salts, preservatives, buffers and the like, as described inREMINGTON'S PHARMACEUTICAL SCIENCES, pp. 1405-12 and 1461-87 (1975) andTHE NATIONAL FORMULARY XIV., 14th Ed. Washington: AmericanPharmaceutical Association (1975). Aqueous carriers include water,alcoholic/aqueous solutions, saline solutions, parenteral vehicles suchas sodium chloride, Ringer's dextrose, etc. Intravenous vehicles includefluid and nutrient replenishers. Preservatives include antimicrobials,anti-oxidants, chelating agents and inert gases. The pH and exactconcentration of the various components of the composition are adjustedaccording to routine skills in the art. See GOODMAN AND GILMAN'S THEPHARMACOLOGICAL BASIS FOR THERAPEUTICS (7th ed.). The peptides of thepresent invention may be present in such pharmaceutical preparations ina concentration of approximately 0.1 to 99.5% by weight, specifically0.5 to 95% by weight, relative to the total mixture. Such pharmaceuticalpreparations may also comprise other pharmaceutically active substancesin addition to the peptides of the present invention. Other methods ofdelivering the peptides to patients will be readily apparent to theskilled artisan.

[0136] Examples of mammalian serine proteases that may exhibitinhibition by the peptides of the present invention include: kallikrein;chymotrypsins A and B; trypsin; elastase; subtilisin; coagulants andprocoagulants, particularly those in active form, including coagulationfactors such as thrombin and factors VIIa, IXa, Xa, XIa, and XIIa;plasmin; proteinase-3; enterokinase; acrosin; cathepsin; urokinase; andtissue plasminogen activator. Examples of conditions associated withincreased serine protease activity include: CPB-induced inflammatoryresponse; post-CPB pulmonary injury; pancreatitis; allergy-inducedprotease release; deep vein thrombosis; thrombocytopenia; rheumatoidarthritis; adult respiratory distress syndrome; chronic inflammatorybowel disease; psoriasis; hyperfibrinolytic hemorrhage; organpreservation; wound healing; and myocardial infarction. Other examplesof the use of the peptides of the present invention are described inU.S. Pat. No. 5,187,153.

[0137] The inhibitors of the present invention may also be used forinhibition of serine protease activity in vitro, for example during thepreparation of cellular extracts to prevent degradation of cellularproteins. For this purpose the inhibitors of the present invention maypreferably be used in a manner analogous to the way that aprotinin, orother known serine protease inhibitors, are used. The use of aprotininas a protease inhibitor for preparation of cellular extracts is wellknown in the art, and aprotinin is sold commercially for this purpose.

[0138] The present invention, thus generally described, will beunderstood more readily by reference to the following examples, whichare provided by way of illustration and are not intended to be limitingof the present invention.

EXAMPLES Example 1

[0139] Expression of Wild-type KPI (−4→57)

[0140] A. Construction of PTW10:KPI

[0141] Plasmid PTW10:KPI is a bacterial expression vector encoding the57 amino acid form of KPI fused to the bacterial phoA signal sequence.The strategy for the construction of PTW10:KPI is shown in FIG. 1.

[0142] Plasmid pcDNAII (Invitrogen, San Diego, Calif.) was digested withPvuII and the larger of the two resulting PvuII fragments (3013 bp) wasisolated. Bacterial expression plasmid pSP26 was digested with MluI andRsrII, and the 409 bp MluI-RsrII fragment containing the pTrp promoterelement and transcription termination signals was isolated byelectrophoresis in a 3% NuSieve Agarose gel (FMC Corp., Rockland, Me.).Plasmid pSP26, containing a heparin-binding EGF-like growth factor(HB-EGF) insert between the NdeI and HindIII sites, is described aspNA28 in Thompson et al., J. Biol. Chem. 269:2541 (1994). Plasmid pSP26was deposited in host E. coli W3110, pSP26 with the American TypeCulture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md., 20852,USA under the conditions specified by the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms (BudapestTreaty). Host E. coli W3110, pSP26 was deposited on May 3, 1995 andgiven Accession No. 69800. Availability of the deposited plasmid is notto be construed as a license to practice the invention in contraventionof the rights granted under the authority of any government inaccordance with its patent laws.

[0143] The ends of the MluI-RsrII fragment were blunted using DNApolymerase Klenow fragment by standard techniques. The blunted fragmentof pSP26 was then ligated into the large PvuII fragment of plasmidpCDNAII, and the ligation mixture was used to transform E. coli strainMC1061. Ampicillin-resistant colonies were selected and used to isolateplasmid pTW10 by standard techniques.

[0144] A synthetic gene was constructed encoding the bacterial phoAsecretory signal sequence fused to the amino terminus of KPI(1→57). Thesynthetic gene contains cohesive ends for NdeI and HindIII, and alsoincorporates restriction endonuclease recognition sites for AgeI, RsrII,AatII and BamHI, as shown in FIG. 2. The synthetic phoA-KPI gene wasconstructed from 6 oligonucleotides of the following sequences (shown5′→3′): 6167: TATGAAACAAAGCACTATTGCACTGGCACTCTTACCGTTACTGTTTACCCCTGTGACAAAAGCCGAGGTGTGCTCTGAA 6169:CTCGGCTTTTGTCACAGGGGTAAACAGTAACGGTAAGAGTGCCAGTGCAA TAGTGCTTTGTTTCATA6165: CAAGCTGAGACCGGTCCGTGCCGTGCAATGATCTCCCGCTGGTACTTTGACGTCACTGAAGGTAAGTGCGCTCCATTCTTT 6166:GCACTTACCTTCAGTGACGTCAAAGTACCAGCGGGAGATCATTGCACGGCACGGACCGGTCTCAGCTTGTTCAGAGCACAC 6168:TACGGCGGTTGCGGCGGCAACCGTAACAACTTTGACACTGAAGAGTACTGCATGGCAGTGTGCGGATCCGCTATTTAAGCT 6164:AGCTTAAATAGCGGATCCGCACACTGCCATGCAGTACTCTTCAGTGTCAAAGTTGTTACGGTTGCCGCCGCAACCGCCGTAAAAGAATGGAGC

[0145] The oligonucleotides were phosphorylated and annealed in pairs:6167+6169, 6165+6166, 6168+6164. In 20 μl T4 DNA Ligase Buffer (NewEngland Biolabs, Beverley, Mass.), 1 μg of each oligonucleotide pair wasincubated with 10 U T4 Polynucleotide Kinase (New England Biolabs) for 1h at 37° C., then heated to 95° C. for 1 minute, and slow-cooled to roomtemperature to allow annealing. All three annealed oligo pairs were thenmixed for ligation to one another in a total volume of 100 μl T4 DNALigase Buffer, and incubated with 400 U T4 DNA Ligase (New EnglandBiolabs) overnight at 15° C. The ligation mixture was extracted with anequal volume of phenol:CHCl₃ (1:1), ethanol-precipitated, resuspended in50 μl Restriction Endonuclease Buffer #4 (New England Biolabs) anddigested with NdeI and HindIII. The annealed, ligated and digestedoligos were then subjected to electrophoresis in a 3% NuSieve Agarosegel, and the 240 bp NdeI-HindIII fragment was excised. This gel-purifiedsynthetic gene was ligated into plasmid pTW10 which had previously beendigested with NdeI and HindIII, and the ligation mixture was used totransform E. coli strain MC1061. Ampicillin-resistant colonies wereselected and used to prepare plasmid pTW10:KPI. This plasmid containsthe phoA-KPI(1→57) fusion protein inserted between the pTrp promoterelement and the transcription termination signals.

[0146] B. Construction of pKPI-61

[0147] The strategy for constructing pKPI-61 is shown in FIG. 3. PlasmidpTW10:KPI was digested with AgeI and HindIII; the resulting 152 bpAgeI-HindIII fragment containing a portion of the KPI synthetic gene wasisolated by preparative gel electrophoresis. An oligonucleotide pair(129+130) encoding the 9 amino-terminal residues of KPI(1→57) and 4amino acids of yeast α-mating factor was phosphorylated and annealed asdescribed above. 129: CTAGATAAAAGAGAGGTGTGCTCTGAACAAGCTGAGA 130:CCGGTCTCAGCTTGTTCAGAGCACACCTCTCTTTTAT

[0148] The annealed oligonucleotides were then ligated to theAgeI-HindIII fragment of the KPI (1→57) synthetic gene. The resulting192 bp XbaI-HindIII synthetic gene (shown in FIG. 4) was purified bypreparative gel electrophoresis, and ligated into plasmid pUC19 whichhad previously been digested with XbaI and HindIII. The ligationproducts were used to transform E. coli strain MC1061.Ampicillin-resistant colonies were picked and used to prepare plasmidPKPI-57 by standard methods. To create a synthetic gene encodingKPI(−4→57), PKPI-57 was digested with XbaI and AgeI and the smallerfragment replaced with annealed oligos 234+235, which encode 4 aminoacid residues of yeast α-mating factor fused a 4 amino acid residueamino-terminal extension of KPI(1→57). 234: CTAGATAAAAGAGAGGTTGTTAGAGAGGTGTGCTCTGAACAAGCTGAGA 235: CCGGTCTCAGCTTGTTCAGAGCACACCTCTCTAACAACCTCTCTTTTAT

[0149] The 4 extra amino acids are encoded in the amyloid β-proteinprecursor/protease nexin-2 (APPI) which contains the KPI domain. Thesynthetic 201 bp XbaI-HindIII fragment encoding KPI(−4→57) in pKPI-61 isshown in FIG. 5.

[0150] C. Assembly of pTW113

[0151] The strategy for the construction of PTW113 is shown in FIG. 6.Plasmid pSP35 was constructed from yeast expression plasmid pYES2(Invitrogen, San Diego, Calif.) as follows. A 267 bp PvuII-XbaI fragmentwas generated by PCR from yeast α-mating factor DNA using oligos 6274and 6273: 6274: GGGGGCAGCTGTATAAACGATTAAAA 6273:GGGGGTCTAGAGATACCCCTTCTTCTTTAG

[0152] This PCR fragment, encoding an 82 amino acid portion of yeastα-mating factor, including the secretory signal peptide and pro-region,was inserted into pYES2 that had been previously digested with PvuII andXbaI. The resulting plasmid is denoted pSP34.

[0153] Two oligonucleotide pairs, 6294+6292 were then ligated to6290+6291, and the resulting 135 bp fragment was purified by gelelectrophoresis. 6294: CTAGATAAAAGAGAGGCTGAGGCTCACGCTGAAGGTACTTTCA CTTC6290: TGACGTCTCTTCTTACTTGGAAGGTCAAGCTGCTAAGGAATTCATCGCTTGGTTGGTCAAAGGTAGAGGTTAAGCTTA 6291:CTAGTAAGCTTAACCTCTACCTTTGACCAACCAAGCGATGAAT TCCTTAGCA 6292:GCTTGACCTTCCAAGTAAGAAGAGACGTCAGAAGTGAAAGTACCTTCAGCGTGAGCCTCAGCCTCTCTTTTAT

[0154] The resulting synthetic fragment was ligated into the XbaI siteof pSP34, resulting in plasmid pSP35. pSP35 was digested with XbaI andHindIII to remove the insert, and ligated with the 201 bp XbaI-HindIIIfragment of pKPI-61, encoding KPI(−4→57). The resulting plasmid pTW113,encodes the 445 bp synthetic gene for the α-factor-KPI(−4→57) fusion.See FIG. 7.

[0155] D. Transformation of yeast with pTW113

[0156]Saccharomyces cerevisiae strain ABL115 was transformed withplasmid pTW113 by electroporation by the method of Becker et al.,Methods Enzymol. 194:182 (1991). An overnight culture of yeast strainABL115 was used to inoculate 200 ml YPD medium. The inoculated culturewas grown with vigorous shaking at 30° C. to an OD₆₀₀ of 1.3-1.5, atwhich time the cells were harvested by centrifugation at 5000 rpm for 5minutes. The cell pellet was resuspended in 200 ml ice-cold water,respun, resuspended in 100 ml ice-cold water, then pelleted again. Thewashed cell pellet was resuspended in 10 ml ice-cold 1M sorbitol,recentrifuged, then resuspended in a final volume of 0.2 ml ice-cold 1Msorbitol. A 40 μl aliquot of cells was placed into the chamber of a cold0.2 cm electroporation cuvette (Invitrogen), along with 100 ng plasmidDNA for pTW113. The cuvette was placed into an Invitrogen ElectroporatorII and pulsed at 1500 V, 25 μF, 100 Ω. Electroporated cells were dilutedwith 0.5 ml 1M sorbitol, and 0.25 ml was spread on an SD agar platecontaining 1M sorbitol. After 3 days' growth at 30° C., individualcolonies were streaked on SD+CAA agar plates.

[0157] E. Induction of pTW113/ABL115, purification of KPI(−4→57)

[0158] Yeast cultures were grown in a rich broth and the galactosepromoter of the KPI expression vector induced with the addition ofgalactose as described by Sherman, Methods Enzymol. 194:3 (1991). Asingle well-isolated colony of pTW113/ABL115 was used to inoculate a 10ml overnight culture in Yeast Batch Medium. The next day, 1 L YeastBatch Medium which had been made 0.2% glucose was inoculated to an OD₆₀₀of 0.1 with the overnight culture. Following 24 hours at 30° C. withvigorous shaking, the 1 L culture was induced by the addition of 20 mlYeast Galactose Feed Medium. Following induction, the culture was fedevery 12 hours with the addition of 20 ml Yeast Galactose Feed Medium.At 48 hours after induction, the yeast broth was harvested bycentrifugation, then adjusted to pH 7.0 with 2M Tris, pH 10. The brothwas subjected to trypsin-Sepharose affinity chromatography, and boundKPI(−4→57) was eluted with 20 mM Tris pH 2.5. See Schilling et al., Gene98:225 (1991). Final purification of KPI(−4→57) was accomplished by HPLCchromatography on a semi-prep Vydac C4 column in a gradient of 20% to35% acetonitrile. The sample was dried and resuspended in PBS at 1-2mg/ml. The amino acid sequence of KPI(−4→57) is shown in FIG. 8.

Example 2

[0159] Recombinant Expression of Site-directed KPI(−4→57) Variants

[0160] Expression vectors for the production of specific variants ofKPI(−4→57) were all constructed using the pTW113 backbone as a startingpoint. For each KPI variant, an expression construct was created byreplacing the 40 bp RsrII-AatII fragment of the synthetic KPI genecontained in pTW113 with a pair of annealed oligonucleotides whichencode specific codons mutated from the wild-type KPI(−4→57) sequence.In the following Examples the convention used for designating the aminosubstituents in the KPI variants indicates first the single letter codefor the amino acid found in wild-type KPI, followed by the position ofthe residue using the numbering convention described supra, followed bythe code for the replacement amino acid. Thus, for example, M15Rindicates that the methionine residue at position 15 is replaced by anarginine.

[0161] A. Construction of pTW6165

[0162] The strategy for constructing pTW6165 is shown in FIG. 9. PlasmidpTW113 was digested with RsrII and AatII, and the larger of the tworesulting fragments was isolated. An oligonucleotide pair (812+813) wasphosphorylated, annealed and gel-purified as described above. 812:GTCCGTGCCGTGCAGCTATCTGGCGCTGGTACTTTGACGT 813:CAAAGTACCAGCGCCAGATAGCTGCACGGCACG

[0163] The annealed oligonucleotides were ligated into the RsrII andAatII-digested pTW113, and the ligation product was used to transform E.coli strain MC1061. Transformed colonies were selected by ampicillinresistance. The resulting plasmid, pTW6165, encodes the 445 bp syntheticgene for the α-factor-KPI(−4→57; M15A, S17W) fusion. See FIG. 10.

[0164] B. Construction of pTW6166, pTW6175, pBG028, pTw6183, pTW6184,pTW6185, pTW6173, pTW6174.

[0165] Construction of the following KPI (−4→57) variants wasaccomplished exactly as outlined for pTW6165. The oligonucleotidesutilized for each construct are denoted below, and the sequences ofannealed oligonucleotide pairs are shown in FIG. 11. FIGS. 12-19 showthe synthetic genes for the α-factor fusions with each KPI(−4→57)variant. pTW6166: KPI (−4→57; M15A, S17Y) - See Figure 12 814:GTCCGTGCCGTGCAGCTATCTACCGCTGGTACTTTGACGT 815:CAAAGTACCAGCGGTAGATAGCTGCACGGCACG pTW6175: KPI (−4→57; M15L, S17F) - SeeFigure 13 867: GTCCGTGCCGTGCATTGATCTTCCGCTGGTACTTTGACGT 868:CAAAGTACCAGCGGAAGATCAATGCACGGCACG pBG028: KPI (−4→57; M15L, S17Y) - SeeFigure 14 1493: GTCCGTGCCGTGCTTTGATCTACCGCTGGTACTTTGACGT 1494:CAAAGTACCAGCGGTAGATCAAAGCACGGCACG pTW6183: KPI (−4→57; I16H, S17F) - SeeFigure 15 925: GTCCGTGCCGTGCAATGCACTTCCGCTGGTACTTTGACGT 926:CAAAGTACCAGCGGAAGTGCATTGCACGGCACG pTW6184: KPI (−4→57; I16H, S17Y) - SeeFigure 16 927: GTCCGTGCCGTGCAATGCACTACCGCTGGTACTTTGACGT 928:CAAAGTACCAGCGGTAGTGCATTGCACGGCACG pTW6185: KPI (−4→57; I16H, S17W) - SeeFigure 17 929: GTCCGTGCCGTGCAATGCACTGGCGCTGGTACTTTGACGT 930:CAAAGTACCAGCGCCAGTGCATTGCACGGCACG pTW6173: KPI (−4→57; M15A, I16H) - SeeFigure 18 863: GTCCGTGCCGTGCAGCTCACTCCCGCTGGTACTTTGACGT 864:CAAAGTACCAGCGGGAGTGAGCTGCACGGCACG pTW6174: KPI (−4→57; M15L, I16H) - SeeFigure 19 865: GTCCGTGCCGTGCATTGCACTCCCGCTGGTACTTTGACGT 866:CAAAGTACCAGCGGGAGTGCAATGCACGGCACG

[0166] C. Transformation of yeast with expression vectors

[0167] Yeast strain ABL115 was transformed by electroporation exactlyaccording to the protocol described for transformation by pTW113.

[0168] D. Induction of transformed yeast strains, purification ofKPI(−4→57) variants.

[0169] Cultures of yeast strains were grown and induced, and recombinantsecreted KPI(−4→57) variants were purified according to the proceduredescribed for KPI(−4→57). The amino acid sequences of KPI(−4→57)variants are shown in FIGS. 20-29.

Example 3

[0170] Identification of KPI (−4→57; M15A, S17F) DD185 by Phage Display

[0171] A. Construction of vector pSP26:Amp:F1

[0172] The construction of pSP26:Amp:F1 is outlined in FIG. 30. VectorpSP26:Amp:F1 contributes the basic plasmid backbone for the constructionof the phage display vector for the phoA:KPI fusion, PDW1 #14.pSP26:Amp:F1 contains a low-copy number origin of replication, theampicillin-resistance gene (Amp) and the F1 origin for production ofsingle-stranded phagemid DNA.

[0173] The ampicillin-resistance gene (Amp) was generated throughpolymerase chain reaction (PCR) amplification from the plasmid genome ofPUC19 using oligonucleotides 176 and 177. 176:GCCATCGATGGTTTCTTAAGCGTCAGGTGGCACTTTTC 177:GCGCCAATTCTTGGTCTACGGGGTCTGACGCTCAGTGGAACGAA

[0174] The PCR amplification of Amp was done according to standardtechniques, using Taq polymerase (Perkin-Elmer Cetus, Norwalk, Conn.).Amplification from plasmid pUC19 with these oligonucleotides yielded afragment of 1159 bp, containing PflMI and ClaI restriction sites. ThePCR product was digested with PflMI and ClaI and purified by agarose gelelectrophoresis in 3% NuSieve Agarose (FMC Corp.). Bacterial expressionvector pSP26 (supra) was digested with PflMI and ClaI and the largervector fragment was purified. The PflMI-ClaI PCR fragment was ligatedinto the previously digested pSP26 containing the Amp gene. The ligationproduct was used to transform E. coli strain MC1061 and colonies wereselected by ampicillin resistance. The resulting plasmid is denotedpSP26:Amp.

[0175] The F1 origin of replication from the mammalian expression vectorpcDNAII (Invitrogen) was isolated in a 692 bp EarI fragment. PlasmidpcDNAII was digested with EarI and the resulting 692 bp fragmentpurified by agarose gel electrophoresis. EarI-NotI adapters were addedto the 692 bp EarI fragment by ligation of two annealed oligonucleotidepairs, 179+180 and 181+182. The oligo pairs were annealed as describedabove. 179: +TL,17GGCCGCTCTTCC 180: AAAGGAAGAGC 181: CTAGAATTGC 182:GGCCGCAATTC

[0176] The oligonucleotide-ligated fragment was then ligated into thesingle NotI site of PSP26:Amp to yield the vector pSP26:Amp:F1.

[0177] B. Construction of vector pgIII

[0178] The construction of pgIII is outlined in FIG. 31. The portion ofthe phage geneIII protein gene contained by the PDW1 #14 phagemid vectorwas originally obtained as a PCR amplification product from vectorm13mp8. A portion of m13mp8 geneIII encoding the carboxyl-terminal 158amino acid residues of the geneIII product was isolated by PCRamplification of m13mp8 nucleotide residues 2307-2781 using PCR oligos6162 and 6160. 6162: GCCGGATCCGCTATTTCCGGTGGTGGCTCTGGTTCC 6160:GCCAAGCTTATTAAGACTCCTTATTACGCAG

[0179] The PCR oligos contain BamHI and HindIII restriction recognitionsites such that PCR from m13mp8 plasmid DNA with the oligo pair yieldeda 490 bp BamHI-HindIII fragment encoding the appropriate portion ofgeneIII. The PCR product was ligated between the BamHI and HindIII siteswithin the polylinker of PUC19 to yield plasmid pgIII.

[0180] C. Construction of pPhoA:KPI:gIII

[0181] Construction of pPhoA:KPI:gIII is outlined in FIG. 32. A portionof the phoA signal sequence and KPI fusion encoded by the phage displayvector PDW1 #14 originates with pPhoA:KPI:gIII. The 237 bp NdeI-HindIIIfragment of pTW10:KPI encoding the entire phoA:KPI (1→57) fusion wasisolated by preparative agarose gel electrophoresis, and insertedbetween the NdeI and HindIII sites of pUC19 to yield plasmid pPhoA:KPI.The 490 bp BamHI-HindIII fragment of pgIII encoding the C-terminalportion of the geneIII product was then isolated and ligated between theBamHI and HindIII sites of pPhoA:KPI to yield vector pPhoa:KPI:gIII. ThepPhoA:KPI:gIII vector encodes a 236 amino acid residue fusion of thephoA signal peptide, KPI (1→57) and the carboxyl-terminal portion of thegeneIII product.

[0182] D. Construction of pLG1

[0183] Construction of pLG1 is illustrated in FIG. 33. The exact geneIIIsequences contained in vector PDW1 #14 originate with phage displayvector pLG1. A modified geneIII segment was generated by PCRamplification of the geneIII region from pgIII using PCRoligonucleotides 6308 and 6305. 6308:AGCTCCGATCTAGGATCCGGTGGTGGCTCTGGTTCCGGT 6305:GCAGCGGCCGTTAAGCTTATTAAGACTCCT

[0184] PCR amplification from pgIII with these oligonucleotides yieldeda 481 bp BamHI-HindIII fragment encoding a geneIII product shortened by3 amino acid residues at the amino-terminal portion of the segment ofthe geneIII fragment encoded by pgIII. A 161 bp NdeI-BamHI fragment wasgenerated by PCR amplification from bacterial expression plasmid pTHW05using oligonucleotides 6306 and 6307. 6306: GATCCTTGTGTCCATATGAAACAAAGC6307: CACGTCGGTCGAGGATCCCTAACCACGGCCTTTAACCAG

[0185] The 161 bp NdeI-BamHI fragment and the 481 bp BamHI-HindIIIfragment were gel-purified, and then ligated in a three-way ligationinto PTW10 which had previously been digested with NdeI and HindIII. Theresulting plasmid pLG1 encodes a phoA signal peptide-insert-geneIIIfusion for phage display purposes.

[0186] E. Construction of pAL51

[0187] Construction of pAL51 is illustrated in FIG. 34. Vector pAL51contains the geneIII sequences of pLG1 which are to be incorporated invector pDW1 #14.

[0188] A 1693 bp fragment of plasmid pBR322 was isolated, extending fromthe BamHI site at nucleotide 375 to the PvuII site at position 2064.Plasmid pLG1 was digested with Asp718I and BamHI, removing an 87 bpfragment. The overhanging Asp718I end was blunted by treatment withKlenow fragment, and the PvuII-BamHI fragment isolated from pBR322 wasligated into this vector, resulting in the insertion of a 1693 bp“stuffer” region between the Asp718I and BamHI sites. The 78 bpNdeI-Asp718I region of the resulting plasmid was removed and replacedwith the annealed oligo pair 6512+6513. 6512:TATGAAACAAAGCACTATTGCACTGGCACTCTTACCGTTACTGTTTACCCCGGTGACCAAAGCCCACGCTGAAG 6513:GTACCTTCAGCGTGGGCTTTGGTCACCGGGGTAAACAGTAACGGTAAGAGTGCCAGTGCAATAGTGCTTTGTTTCA

[0189] The newly created 74 bp NdeI-Asp718I fragment encodes the phoAsignal peptide, and contains a BstEII cloning site. The resultingplasmid is denoted pAL51.

[0190] F. Construction of pAL53

[0191] Construction of pAL53 is outlined in FIG. 35. Plasmid pAL53contributes most of the vector sequence of pDW1 #14, including the basicvector backbone with Amp gene, F1 origin, low copy number origin ofreplication, geneIII segment, phoA promotor and phoA signal sequence.

[0192] Plasmid pAL51 was digested with NdeI and HindIII and theresulting 2248 bp NdeI-HindIII fragment encoding the phoA signalpeptide, stuffer region and geneIII region was isolated by preparativeagarose gel electrophoresis. The NdeI-HindIII fragment was ligated intoplasmid pSP26:Amp:F1 between the NdeI and HindIII sites, resulting inplasmid pAL52.

[0193] The phoA promoter region and signal peptide was generated byamplification of a portion of the E. coli genome by PCR, usingoligonucleotide primers 405 and 406. 405: CCGGACGCGTGGAGATTATCGTCACTG406: GCTTTGGTCACCGGGGTAAACAGTAACGG

[0194] The resulting PCR product is a 332 bp MluI-BstEII fragment whichcontains the phoA promoter region and signal peptide sequence. Thisfragment was used to replace the 148 bp MluI-BstEII segment of PAL52,resulting in vector pAL53.

[0195] G. Construction of pSP26:Amp:F1:PhoA:KPI:gIII

[0196] Construction of pSP26:Amp:F1:PhoA:KPI:gIII is illustrated in FIG.36. This particular vector is the source of the KPI coding sequencefound in vector pDW1 #14. Plasmid pPhoa:KPI:gIII was digested with NdeIand HindIII, and the resulting 714 bp NdeI-HindIII fragment waspurified, and then inserted into vector pSP26:Amp:F1 between the NdeIand HindIII sites. The resulting plasmid is denotedpSP26:Amp:F1:PhoA:KPI:gIII.

[0197] H. Construction of pDW1 #14

[0198] Construction of pDW1 #14 is illustrated in FIG. 37. The sequencesencoding KPI were amplified from plasmid pSP26:Amp:F1:PhoA:KPI:gIII byPCR, using oligonucleotide primers 424 and 425. 424:CTGTTTACCCCGGTGACCAAAGCCGAGGTGTGCTCTGAACAA 425:AATAGCGGATCCGCACACTGCCATGCAGTACTCTTC

[0199] The resulting 172 bp BstEII-BamHI fragment encodes most of KPI(1→55). This fragment was used to replace the stuffer region in pAL53between the BstEII and BamHI sites. The resulting plasmid, PDW1 #14, isthe parent KPI phage display vector for preparation of randomized KPIphage libraries. The coding region for the phoA-KPI (1→55)-geneIIIfusion is shown in FIG. 38.

[0200] I. Construction of pDW1 14-2

[0201] Construction of pDW1 14-2 is illustrated in FIG. 39. The firststep in the construction of the KPI phage libraries in pDW1 #14 was thereplacement of the AgeI-BamHI fragment within the KPI coding sequencewith a stuffer fragment. This greatly aids in preparation of randomizedKPI libraries which are substantially free of contamination of phagemidgenomes encoding wild-type KPI sequence.

[0202] Plasmid pDW1 #14 was digested with AgeI and BamHI, and the 135 bpAgeI-BamHI fragment encoding KPI was discarded. A stuffer fragment wascreated by PCR amplification of a portion of the PBR322 Tet gene,extending from the BamHI site at nucleotide 375 to nucleotide 1284,using oligo primers 266 and 252. 266:GCTTTAAACCGGTAGGTGGCCCGGCTCCATGCACC 252:CGAATTCACCGGTGTCATCCTCGGCACCGTCACCCT

[0203] The resulting 894 bp AgeI-BamHI stuffer fragment was theninserted into the AgeI/BamHI-digested pDW1 #14 to yield the phagemidvector pDW1 14-2. This vector was the starting point for construction ofthe randomized KPI libraries.

[0204] J. Construction of KPI Library 16-19

[0205] Construction of KPI Library 16-19 is outlined in FIG. 40. Library16-19 was constructed to display KPI-geneIII fusions in which amino acidpositions Ala¹⁴, Met¹⁵, Ile¹⁶ and Ser¹⁷ are randomized. For preparationof the library, plasmid pDW1 14-2 was digested with AgeI and BamHI toremove the stuffer region, and the resulting vector was purified bypreparative agarose gel electrophoresis. Plasmid PDW1 #14 was used astemplate in a PCR amplification of the KPI region extending from theAgeI site to the BamHI site. The oligonucleotide primers used were 544and 551. 544: GGGCTGAGACCGGTCCGTGCCGT(NNS)₄CGCTGGTACTTTGACGTC 551:GGAATAGCGGATCCGCACACTGCCATGCAG

[0206] Oligonucleotide primer 544 contains four randomized codons of thesequence NNS, where N represents equal mixtures of A/G/C/T and S anequal mixture of G or C. Each NNS codon thus encodes all 20 amino acidsplus a single possible stop codon, in 32 different DNA sequences. PCRamplification from the wild-type KPI gene resulted in the production ofa mixture of 135 bp AgeI-BamHI fragments all containing differentsequences in the randomized region. The PCR product was purified bypreparative agarose gel electrophoresis and ligated into the AgeI/BamHIdigested PDW1 14-2 vector. The ligation mixture was used to transform E.coli Top10F¹ cells (Invitrogen) by electroporation according to themanufacturer's directions. The resulting Library 16-19 containedapproximately 400,000 independent clones. The potential size of thelibrary, based upon the degeneracy of the priming PCR oligo #544 was1,048,576 members. The expression unit encoded by the members of Library16-19 is shown in FIG. 41.

[0207] K. Selection of Library 16-19 with human plasma kallikrein

[0208] KPI phage were prepared and amplified by infecting transformedcells with M13K07 helper phage as described by Matthews et al., Science260:1113 (1993). Human plasma kallikrein (Enzyme Research Laboratories,South Bend, Ind.), was coupled to Sepharose 6B resin. Prior to phagebinding, the immobilized kallikrein resin was washed three times with0.5 ml assay buffer (AB=100 mM Tris-HCl, pH 7.5, 0.5M NaCl, 5 mM each ofKC1, CaCl₂, MgCl₂, 0.1% gelatin, and 0.05% Triton X-100). Approximately5×10⁹ phage particles of the amplified Library 16-19 in PBS, pH 7.5,containing 300 mM NaCl and 0.1% gelatin, were bound to 50 μl kallikreinresin containing 15 pmoles of active human plasma kallikrein in a totalvolume of 250 μl. Phage were allowed to bind for 4 h at roomtemperature, with rocking. Unbound phage were removed by washing thekallikrein resin three times in 0.5 ml AB. Bound phage were elutedsequentially by successive 5 minute washes: 0.5 ml 50 mM sodium citrate,pH 6.0, 150 mM NaCl; 0.5 ml 50 mM sodium citrate, pH 4.0, 150 mM NaCl;and 0.5 ml 50 mM glycine, pH 2.0, 150 mM NaCl. Eluted phage wereneutralized immediately and phagemids from the pH 2.0 elution weretitered and amplified for reselection. After three rounds of selectionon kallikrein-Sepharose, phagemid DNA was isolated from 22 individualcolonies and subjected to DNA sequence analysis.

[0209] The most frequently occurring randomized KPI region encoded:Ala¹⁴-Ala¹⁵-Ile¹⁶-Phe¹⁷. The phoA-KPI-geneIII region encoded by thisclass of selected KPI phage is shown in FIG. 42. The KPI variant encodedby these phagemids is denoted KPI (1→55; M15A, S17F).

[0210] L. Construction of pDD185 KPI (−4→57; M15A, S17F)

[0211]FIG. 43 outlines the construction of pDD185 KPI (−4→57; M15A,S17F). The sequences encoding KPI (1→55; M15A, S17F) were moved from onephagemid vector, pDW1 (16-19) 185, to the yeast expression vector sothat the KPI variant could be purified and tested.

[0212] Plasmid pTW113 encoding wild-type KPI (−4→57) was digested withAgeI and BamHI and the 135 bp AgeI-BamHI fragment was discarded. The 135bp AgeI-BamHI fragment of pDW1 (16-19) 185 was isolated and ligated intothe yeast vector to yield plasmid pDD185, encoding α-factor fused to KPI(−4→57; M15A, S17F). See FIG. 44.

[0213] M. Purification of KPI (−4→57; M15A, S17F) pDD185

[0214] Transformation of yeast strain ABL115 with pDD185, induction ofyeast cultures, and purification of KPI (−4→57; M15A, S17F) pDD185 wasaccomplished as described for the other KPI variants.

[0215] N. Construction of KPI Library 6—M15A, with residues 14, 16-18random.

[0216] Library 6 was constructed to display KPI-geneIII fusions in whichamino acid positions Ala¹⁴, Ile¹⁶, Ser¹⁷ and Arg¹⁸ are randomized, butposition 15 was held constant as Ala. For preparation of the library,plasmid pDW1 #14 was used as template in a PCR amplification of the KPIregion extending from the AgeI site to the BamHI site. Theoligonucleotide primers used were 551 and 1003. 1003:GCTGAGACCGGTCCGTGCCGTNNSGCA(NNS)₃TGGTACTTTGACGTC 551:GGAATAGCGGATCCGCACACTGCCATGCAG

[0217] Oligonucleotide primer 1003 contained four randomized codons ofthe sequence NNS, where N represents equal mixtures of A/G/C/T and S anequal mixture of G or C. Each NNS codon thus encodes all 20 amino acidsplus a single possible stop, in 32 different DNA sequences. PCRamplification from the wild-type KPI gene resulted in the production ofa mixture of 135 bp AgeI-BamHI fragments all containing differentsequences in the randomized region. The PCR product was phenolextracted, ethanol precipitated, digested with BamHI and purified bypreparative agarose gel electrophoresis. Plasmid pDW1 14-2 was digestedwith BamHI, phenol extracted and ethanol precipitated. The insert wasligated at high molar ratio to the vector which was then digested withAgeI to remove the stuffer region. The vector containing the insert waspurified by agarose gel electrophoresis and recircularized. Theresulting library contains approximately 5×10⁶ independent clones.

[0218] O. Construction of KPI Library 7—residues 14-18 random.

[0219] Library 7 was constructed to display KPI-geneIII fusions in whichamino acid positions Ala¹⁴, Met¹⁵, Ile¹⁶, Ser¹⁷ and Arg¹⁸ arerandomized. For preparation of the library, plasmid pDW1 #14 was used astemplate in a PCR amplification of the KPI region extending from theAgeI site to the BamHI site. The oligonucleotide primers used were 551and 1179. 1179: GCTGAGACCGGTCCGTGCCGT(NNS)₅TGGTACTTTGACGTC 551:GGAATAGCGGATCCGCACACTGCCATGCAG

[0220] Oligonucleotide primer 1179 contains five randomized codons ofthe sequence NNS, where N represents equal mixtures of A/G/C/T and S anequal mixture of G or C. Each NNS codon thus encoded all 20 amino acidsplus a single possible stop, in 32 different DNA sequences. PCRamplification from the wild-type KPI gene resulted in the production ofa mixture of 135 bp AgeI-BamHI fragments all containing differentsequences in the randomized region. The PCR product was phenolextracted, ethanol precipitated, digested with BamHI and purified bypreparative agarose gel electrophoresis. Plasmid pDW1 14-2 was digestedwith BamHI, phenol extracted and ethanol precipitated. The insert wasligated at high molar ratio to the vector which was then digested withAgeI to remove the stuffer region. The vector containing the insert waspurified by agarose gel electrophoresis and recircularized. Theresulting library contains approximately 1×10⁷ independent clones.

[0221] P. Selection of Libraries 6 & 7 with human factor XIIa

[0222] KPI phage were prepared and amplified by infecting transformedcells with M13K07 helper phage (Matthews and Wells, 1993). Human factorXIIa (Enzyme Research Laboratories, South Bend, Ind.), was biotinylatedas follows. Factor XIIa (0.5 mg) in 5 mM sodium acetate pH 8.3 wasincubated with Biotin Ester (Zymed) at room temperature for 1.5 h, thenbuffer-exchanged into assay buffer (AB). Approximately 1×10¹⁰ phageparticles of each amplified Library 6 or 7 in PBS, pH 7.5, containing300 mM NaCl and 0.1% gelatin, were incubated with 50 pmoles of activebiotinylated human factor XIIa in a total volume of 200 μl. Phage wereallowed to bind for 2 h at room temperature, with rocking. Following thebinding period, 100 μl Strepavidin Magnetic Particles (BoehringerMannheim) were added to the mixture and incubated at room temperaturefor 30 minutes. Separation of magnetic particles from the supernatantand wash/elution buffers was carried out using MPC-E-1Neodymium-iron-boron permanent magnets (Dynal). Unbound phage wereremoved by washing the magnetically bound biotinylated XIIa-phagecomplexes three times with 0.5 ml AB. Bound phage were elutedsequentially by successive 5 minute washes: 0.5 ml 50 mM sodium citrate,pH 6.0, 150 mM NaCl; 0.5 ml 50 mM sodium citrate, pH 4.0, 150 mM NaCl;and 0.5 ml 50 mM glycine, pH 2.0, 150 mM NaCl. Eluted phage wereneutralized immediately and phagemids from the pH 2.0 elution weretitered and amplified for reselection. After 3 or 4 rounds of selectionwith factor XIIa, phagemid DNA was isolated from individual colonies andsubjected to DNA sequence analysis.

[0223] Sequences in the randomized regions were compared with oneanother to identify consensus sequences appearing more than once. FromLibrary 6 a phagemid was identified which encoded M15L, S17Y, R18H. FromLibrary 7 a phagemid was identified which encoded M15A, S17Y, R18H.

[0224] Q. Construction of pBG015 KPI (−4→57; M15L, S17Y, R18H), pBG022(−4→57; M15A, S17Y, R18H)

[0225] The sequences encoding KPI (1→55; M15L, S17Y, R18H) and KPI(1→55; M17A, S17Y, R18H) were moved from the phagemid vectors to theyeast expression vector so that the KPI variant could be purified andtested.

[0226] Plasmid pTW113 encoding wild-type KPI (−4→57) was digested withAgeI and BamHI and the 135 bp AgeI-BamHI fragment was discarded. The 135bp AgeI-BamHI fragment of the phagemid vectors were isolated and ligatedinto the yeast vector to yield plasmids pBG015 and pBG022, encodingalpha-factor fused to KPI (−4→57; M15L, S17Y, R18H), and KPI (−4→57;M15A, S17Y, R18H), respectively.

[0227] R. Construction of pBG029 KPI (−4→57, T9V, M15L, S17Y, R18H)

[0228] Plasmid pBG015 was digested with XbaI and RsrII, and the largerof the two resulting fragments was isolated. An oligonucleotide pair(1593+1642) was phosphorylated, annealed and gel-purified as describedpreviously. 1593: CTAGATAAAAGAGAGGTTGTTAGAGAGGTGTGCTCTGAACAAGC TGAGGTTG1642: GACCAACCTCAGCTTGTTCAGAGCACACCTCTCTAA CAACCTCTCTTTTAT

[0229] The annealed oligonucleotides were ligated into the XbaI andRsrII-digested pBG015, and the ligation product was used to transform E.coli strain MC1061 to ampicillin resistance. The resulting plasmidpBG029, encodes the 445 bp synthetic gene for the alpha-factor-KPI(−4→57; T9V, M15L, S17F, R18H) fusion.

[0230] S. Construction of pBG033 KPI (−4→57; T9V, M15A, S17Y, R18H)

[0231] Plasmid pBG022 was digested with XbaI and RsrII, and the largerof the two resulting fragments was isolated. An oligonucleotide pair(1593+1642) was phosphorylated, annealed and gel-purified as describedpreviously. The annealed oligonucleotides were ligated into the XbaI andRsrII-digested pBG022, and the ligation product was used to transform E.coli strain MC1061 to ampicillin resistance. The resulting plasmidpBG033, encodes the 445 bp synthetic gene for the alpha-factor-KPI(−4→57; T9V, M15A, S17F, R18H) fusion.

[0232] T. Selection of Library 16-19 with human factor Xa

[0233] KPI phage were prepared and amplified by infecting transformedcells with M13K07 helper phage (Matthews and Wells, 1993). Human factorXa (Haematologic Technologies, Inc., Essex Junction, Vt.) was coupled toSepharose 6B resin. Prior to phage binding, the immobilized Xa resin waswashed three times with 0.5 ml assay buffer (AB=100 mM Tris-HCl, pH 7.5,0.5M NaCl, 5 mM each of KCl, CaCl₂, MgCl₂, 0.1% gelatin, and 0.05%Triton X-100). Approximately 4×10¹⁰ phage particles of the amplifiedLibrary 16-19 in PBS, pH 7.5, containing 300 mM NaCl and 0.1% gelatin,were bound to 50 μl Xa resin in a total volume of 250 μl. Phage wereallowed to bind for 4 h at room temperature, with rocking. Unbound phagewere removed by washing the Xa resin three times in 0.5 ml AB. Boundphage were eluted sequentially by successive 5 minute washes: 0.5 ml 50mM sodium citrate, pH 6.0, 150 mM NaCl; 0.5 ml 50 mM sodium citrate, pH4.0 150 mM NaCl; and 0.5 ml 50 mM glycine, pH 2.0, 150 mM NaCl. Elutedphage were neutralized immediately and phagemids from the pH 2.0 elutionwere titered and amplified for reselection. After three rounds ofselection on Xa-Sepharose, phagemid DNA was isolated and subjected toDNA sequence analysis.

[0234] Sequences in the randomized Ala¹⁴-Ser¹⁷ region were compared withone another to identify consensus sequences appearing more than once. Aphagemid was identified which encoded KPI (1→55; M15L, I16F, S17K).

[0235] U. Construction of pDD131 KPI (−4→57; M15L, I16F, S17K)

[0236] The sequences encoding KPI (1→55; M15L, I16F, S17K) were movedfrom the phagemid vector to the yeast expression vector so that the KPIvariant could be purified and tested.

[0237] Plasmid pTW113 encoding wild-type KPI (−4→57) was digested withAgeI and BamHI and the 135 bp AgeI-BamHI fragment was discarded. The 135bp AgeI-BamHI fragment of the phagemid vector was isolated and ligatedinto the yeast vector to yield plasmid pDD131, encoding alpha-factorfused to KPI (−4→57; M15L, I16F, S17K).

[0238] V. Construction of pDD134 KPI (−4→57; M15L, I16F, S17K, G37Y)

[0239] Plasmid pDD131 was digested with AatI and BamHI, and the largerof the two resulting fragments was isolated. An oligonucleotide pair(738+739) was phosphorylated, annealed and gel-purified as describedpreviously. 738: CACTGAAGGTAAGTGCGCTCCATTCTTTTACGGCGGTTGCTACGGCAACCGTAACAACTTTGACACTGAAGAGTACTGCATGGCAGTGTG CG 739:GATCCGCACACTGCCATGCAGTACTCTTCAGTGTCAAAGTTGTTACGGTTGCCGTAGCAACCGCCGTAAAAGAATGGAGCGCACTTACCT TCAGTGACGT

[0240] The annealed oligonucleotides were ligated into the AatI andBamHI-digested pDD131, and the ligation product was used to transform E.coli strain MC1061 to ampicillin resistance. The resulting plasmidpDD134, encodes the 445 bp synthetic gene for the alpha-factor-KPI(−4→57; M15L, I16F, S17K, G37Y) fusion.

[0241] W. Construction of pDD135 KPI (−4→57; M15L, I16F, S17K, G37L)

[0242] Plasmid pDD131 was digested with AatII and BamHI, and the largerof the two resulting fragments was isolated. An oligonucleotide pair(724+725) was phosphorylated, annealed and gel-purified as describedpreviously. 738: CACTGAAGGTAAGTGCGCTCCATTCTTTTACGGCGGTTGCTACGGCAACCGTAACAACTTTGACACTGAAGAGTACTGCATGGCAGTGTG CG 739:GATCCGCACACTGCCATGCAGTACTCTTCAGTGTCAAAGTTGTTACGGTTGCCGTAGCAACCGCCGTAAAAGAATGGAGCGCACTTACCT TCAGTGACGT

[0243] The annealed oligonucleotides were ligated into the AatII andBamHI-digested pDD131, and the ligation product was used to transform E.coli strain MC1061 to ampicillin resistance. The resulting plasmidpDD135, encodes the 445 bp synthetic gene for the alpha-factor-KPI(−4→57; M15L, I16F, S17K, G37L) fusion.

Example 4

[0244] Kinetic Analysis of KPI(−4→57) Variants

[0245] The concentrations of active human plasma kallikrein, factorXIIa, and trypsin were determined by titration with p-nitrophenylp′-guanidinobenzoate as described by Bender et al., supra, and Chase etal., Biochem. Biophys. Res. Commun. 29:508 (1967). Accurateconcentrations of active KPI (−4→57) inhibitors were determined bytitration of the activity of a known amount of active-site-titratedtrypsin. For testing against kallikrein and trypsin, each KPI(−4→57)variant (0.5 to 100 nM) was incubated with protease in low-binding96-well microtiter plates at 30° C. for 15-25 min, in 100 mM Tris-HCl,pH 7.5, with 500 mM NaCl, 5 mM KCl, 5 mM CaCl2, 5 mM MgCl2, 0.1% Difcogelatin, and 0.05% Triton X-100. Chromogenic synthetic substrate wasthen be added, and initial rates at 30° C. recorded by the SOFTmaxkinetics program via a THERMOmax microplate reader (Molecular DevicesCorp., Menlo Park, Calif.). The substrates used were N-α-benzoyl-L-Argp-nitroanilide (1 mM) for trypsin (20 nM), and N-benzoyl-Pro-Phe-Argp-nitroanilide (0.3 mM) for plasma kallikrein (1 nM). The Enzfitter(Elsevier) program was used both to plot fractional activity (i.e.,activity with inhibitor, divided by activity without inhibitor), a,versus total concentration of inhibitor, I_(t), and to calculate thedissociation constant of the inhibitor (K_(i)) by fitting the curve tothe following equation:$a = {1 - \frac{\lbrack E\rbrack_{t} + \lbrack I\rbrack_{t} + K_{i} - \sqrt{\left( {\lbrack E\rbrack_{t} + \lbrack I\rbrack_{t} + K_{i}} \right)^{2} - {{4\lbrack E\rbrack}_{t}\lbrack I\rbrack}_{t}}}{{2\lbrack E\rbrack}_{t}}}$

[0246] The K_(i)s determined for purified KPI variants are shown in FIG.45. The most potent variant, KPI (−4→57; M15A, S17F) DD185 is 115-foldmore potent as a human kallikrein inhibitor than wild-type KPI (−4→57).The least potent variant, KPI (−4→57; I16H, S17W) TW6185 is still35-fold more potent than wild-type KPI.

[0247] For testing against factor XIIa, essentially the same reactionconditions were used, except that the substrate wasN-benzoyl-Ile—Glu—Gly—Arg p-nitroaniline hydrochloride and its methylester (obtained from Pharmacia Hepar, Franklin, Ohio), and corn trypsininhibitor (Enzyme Research Laboratories, South Bend, Ind.) was used as acontrol inhibitor. Factor XIIa was also obtained from Enzyme ResearchLaboratories.

[0248] Various data for inhibition of the serine proteases of interestkallikrein, plasmin, and factors Xa, XIa, and XIIa by a series of KPIvariants are given in FIG. 46. The results indicate that KPI variantscan be produced that can bind to and preferably inhibit the activity ofserine proteases. The results also indicate that the peptides of theinvention may exhibit the preferable more potent and specific inhibitionof one or more serine proteases of interest.

Example 5

[0249] Effect of KPI Variant KPI185-1 on Postoperative Bleeding

[0250] A randomized, double-blinded study using an acute porcinecardiopulmonary bypass (CPB) model was used to investigate the effect ofKPI185-1 on postoperative bleeding. Sixteen pigs (55-65 kg) underwent 60minutes of hypothermic (28° C.) open-chest CPB with 30 minutes ofcardioplegic cardiac arrest. Pigs were randomized against a controlsolution of physiological saline (NS; n=8) or KPI-185 (n=8) groups.During aortic cross-clamping, the tricuspid valve was inspected throughan atriotomy which was subsequently repaired. Following reversal ofheparin with protamine, dilateral thoracostomy tubes were placed andshed blood collected for 3 hours. Shed blood volume and hemoglobin (Hgb)loss were calculated from total chest tube output and residualintrathoracic blood at time of sacrifice.

[0251] Total blood loss was significantly reduced in the KPI185-1 group(245.75±66.24 ml vs. 344.25±63.97 ml, p=0.009). In addition, there was amarked reduction in total Hgb loss in the treatment group (13.59±4.26 gmvs. 23.61±4.69 gm, p=0.0005). Thoracostomy drainage Hgb wassignificantly increased at 30 and 60 minutes in the control group[6.89±1.44 vs. 4.41±1.45 gm/dl (p=0.004) and 7.6±1.03 vs. 5.26±1.04gm/dl (p=0.0002), respectively]. Preoperative and post-CPB hematocritswere not statistically different between the groups. These results areshown in graphical form in FIGS. 47-50.

[0252] The invention has been disclosed broadly and illustrated inreference to representative embodiments described above. Those skilledin the art will recognize that various modifications can be made to thepresent invention without departing from the spirit and scope thereof.

1 228 57 amino acids amino acid single linear protein 1 Xaa Val Cys SerGlu Gln Ala Glu Xaa Gly Xaa Cys Arg Ala Xaa Xaa 1 5 10 15 Xaa Xaa TrpTyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Xaa 20 25 30 Tyr Gly GlyCys Xaa Xaa Xaa Xaa Asn Asn Phe Asp Thr Glu Glu Tyr 35 40 45 Cys Met AlaVal Cys Gly Ser Ala Ile 50 55 5 amino acids amino acid single linearprotein 2 Glu Val Val Arg Glu 1 5 57 amino acids amino acid singlelinear protein 3 Xaa Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg AlaXaa Xaa 1 5 10 15 Xaa Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys AlaPro Phe Phe 20 25 30 Tyr Gly Gly Cys Xaa Gly Asn Arg Asn Asn Phe Asp ThrGlu Glu Tyr 35 40 45 Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 61 aminoacids amino acid single linear protein 4 Glu Val Val Arg Glu Val Cys SerGlu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Xaa Xaa Xaa Arg TrpTyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly GlyCys Xaa Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met AlaVal Cys Gly Ser Ala Ile 50 55 60 57 amino acids amino acid single linearprotein 5 Xaa Val Cys Ser Glu Gln Ala Glu Xaa Gly Pro Cys Arg Ala XaaXaa 1 5 10 15 Xaa Xaa Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys AlaPro Phe 20 25 30 Phe Tyr Gly Gly Cys Xaa Gly Asn Arg Asn Asn Phe Asp ThrGlu Glu 35 40 45 Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 59 aminoacids amino acid single linear protein 6 Val Arg Glu Val Cys Ser Glu GlnAla Glu Thr Gly Pro Cys Arg Ala 1 5 10 15 Met Ile Ser Arg Trp Tyr PheAsp Val Thr Glu Gly Lys Cys Ala Pro 20 25 30 Phe Phe Tyr Gly Gly Cys GlyGly Asn Arg Asn Asn Phe Asp Thr Glu 35 40 45 Glu Tyr Cys Met Ala Val CysGly Ser Ala Ile 50 55 58 amino acids amino acid single linear protein 7Arg Pro Asp Phe Cys Leu Glu Pro Pro Tyr Thr Gly Pro Cys Lys Ala 1 5 1015 Arg Ile Ile Arg Tyr Phe Tyr Asn Ala Lys Ala Gly Leu Cys Gln Thr 20 2530 Phe Val Tyr Gly Gly Cys Arg Ala Lys Arg Asn Asn Phe Lys Ser Ala 35 4045 Glu Asp Cys Met Arg Thr Cys Gly Gly Ala 50 55 4 amino acids aminoacid single linear protein 8 Glu Val Val Arg 1 79 base pairs nucleicacid single linear DNA (genomic) 9 TATGAAACAA AGCACTATTG CACTGGCACTCTTACCGTTA CTGTTTACCC CTGTGACAAA 60 AGCCGAGGTG TGCTCTGAA 79 67 basepairs nucleic acid single linear DNA (genomic) 10 CTCGGCTTTT GTCACAGGGGTAAACAGTAA CGGTAAGAGT GCCAGTGCAA TAGTGCTTTG 60 TTTCATA 67 81 base pairsnucleic acid single linear DNA (genomic) 11 CAAGCTGAGA CCGGTCCGTGCCGTGCAATG ATCTCCCGCT GGTACTTTGA CGTCACTGAA 60 GGTAAGTGCG CTCCATTCTT T81 81 base pairs nucleic acid single linear DNA (genomic) 12 GCACTTACCTTCAGTGACGT CAAAGTACCA GCGGGAGATC ATTGCACGGC ACGGACCGGT 60 CTCAGCTTGTTCAGAGCACA C 81 81 base pairs nucleic acid single linear DNA (genomic)13 TACGGCGGTT GCGGCGGCAA CCGTAACAAC TTTGACACTG AAGAGTACTG CATGGCAGTG 60TGCGGATCCG CTATTTAAGC T 81 93 base pairs nucleic acid single linear DNA(genomic) 14 AGCTTAAATA GCGGATCCGC ACACTGCCAT GCAGTACTCT TCAGTGTCAAAGTTGTTACG 60 GTTGCCGCCG CAACCGCCGT AAAAGAATGG AGC 93 37 base pairsnucleic acid single linear DNA (genomic) 15 CTAGATAAAA GAGAGGTGTGCTCTGAACAA GCTGAGA 37 37 base pairs nucleic acid single linear DNA(genomic) 16 CCGGTCTCAG CTTGTTCAGA GCACACCTCT CTTTTAT 37 49 base pairsnucleic acid single linear DNA (genomic) 17 CTAGATAAAA GAGAGGTTGTTAGAGAGGTG TGCTCTGAAC AAGCTGAGA 49 49 base pairs nucleic acid singlelinear DNA (genomic) 18 CCGGTCTCAG CTTGTTCAGA GCACACCTCT CTAACAACCTCTCTTTTAT 49 26 base pairs nucleic acid single linear DNA (genomic) 19GGGGGCAGCT GTATAAACGA TTAAAA 26 30 base pairs nucleic acid single linearDNA (genomic) 20 GGGGGTCTAG AGATACCCCT TCTTCTTTAG 30 47 base pairsnucleic acid single linear DNA (genomic) 21 CTAGATAAAA GAGAGGCTGAGGCTCACGCT GAAGGTACTT TCACTTC 47 78 base pairs nucleic acid singlelinear DNA (genomic) 22 TGACGTCTCT TCTTACTTGG AAGGTCAAGC TGCTAAGGAATTCATCGCTT GGTTGGTCAA 60 AGGTAGAGGT TAAGCTTA 78 52 base pairs nucleicacid single linear DNA (genomic) 23 CTAGTAAGCT TAACCTCTAC CTTTGACCAACCAAGCGATG AATTCCTTAG CA 52 73 base pairs nucleic acid single linear DNA(genomic) 24 GCTTGACCTT CCAAGTAAGA AGAGACGTCA GAAGTGAAAG TACCTTCAGCGTGAGCCTCA 60 GCCTCTCTTT TAT 73 40 base pairs nucleic acid single linearDNA (genomic) 25 GTCCGTGCCG TGCAGCTATC TGGCGCTGGT ACTTTGACGT 40 33 basepairs nucleic acid single linear DNA (genomic) 26 CAAAGTACCA GCGCCAGATAGCTGCACGGC ACG 33 40 base pairs nucleic acid single linear DNA (genomic)27 GTCCGTGCCG TGCAGCTATC TACCGCTGGT ACTTTGACGT 40 33 base pairs nucleicacid single linear DNA (genomic) 28 CAAAGTACCA GCGGTAGATA GCTGCACGGC ACG33 40 base pairs nucleic acid single linear DNA (genomic) 29 GTCCGTGCCGTGCATTGATC TTCCGCTGGT ACTTTGACGT 40 33 base pairs nucleic acid singlelinear DNA (genomic) 30 CAAAGTACCA GCGGAAGATC AATGCACGGC ACG 33 40 basepairs nucleic acid single linear DNA (genomic) 31 GTCCGTGCCG TGCTTTGATCTACCGCTGGT ACTTTGACGT 40 33 base pairs nucleic acid single linear DNA(genomic) 32 CAAAGTACCA GCGGTAGATC AAAGCACGGC ACG 33 40 base pairsnucleic acid single linear DNA (genomic) 33 GTCCGTGCCG TGCAATGCACTTCCGCTGGT ACTTTGACGT 40 33 base pairs nucleic acid single linear DNA(genomic) 34 CAAAGTACCA GCGGAAGTGC ATTGCACGGC ACG 33 40 base pairsnucleic acid single linear DNA (genomic) 35 GTCCGTGCCG TGCAATGCACTACCGCTGGT ACTTTGACGT 40 33 base pairs nucleic acid single linear DNA(genomic) 36 CAAAGTACCA GCGGTAGTGC ATTGCACGGC ACG 33 40 base pairsnucleic acid single linear DNA (genomic) 37 GTCCGTGCCG TGCAATGCACTGGCGCTGGT ACTTTGACGT 40 33 base pairs nucleic acid single linear DNA(genomic) 38 CAAAGTACCA GCGCCAGTGC ATTGCACGGC ACG 33 40 base pairsnucleic acid single linear DNA (genomic) 39 GTCCGTGCCG TGCAGCTCACTCCCGCTGGT ACTTTGACGT 40 33 base pairs nucleic acid single linear DNA(genomic) 40 CAAAGTACCA GCGGGAGTGA GCTGCACGGC ACG 33 40 base pairsnucleic acid single linear DNA (genomic) 41 GTCCGTGCCG TGCATTGCACTCCCGCTGGT ACTTTGACGT 40 33 base pairs nucleic acid single linear DNA(genomic) 42 CAAAGTACCA GCGGGAGTGC AATGCACGGC ACG 33 38 base pairsnucleic acid single linear DNA (genomic) 43 GCCATCGATG GTTTCTTAAGCGTCAGGTGG CACTTTTC 38 44 base pairs nucleic acid single linear DNA(genomic) 44 GCGCCAATTC TTGGTCTACG GGGTCTGACG CTCAGTGGAA CGAA 44 12 basepairs nucleic acid single linear DNA (genomic) 45 GGCCGCTCTT CC 12 11base pairs nucleic acid single linear DNA (genomic) 46 AAAGGAAGAG C 1110 base pairs nucleic acid single linear DNA (genomic) 47 CTAGAATTGC 1011 base pairs nucleic acid single linear DNA (genomic) 48 GGCCGCAATT C11 36 base pairs nucleic acid single linear DNA (genomic) 49 GCCGGATCCGCTATTTCCGG TGGTGGCTCT GGTTCC 36 31 base pairs nucleic acid single linearDNA (genomic) 50 GCCAAGCTTA TTAAGACTCC TTATTACGCA G 31 39 base pairsnucleic acid single linear DNA (genomic) 51 AGCTCCGATC TAGGATCCGGTGGTGGCTCT GGTTCCGGT 39 30 base pairs nucleic acid single linear DNA(genomic) 52 GCAGCGGCCG TTAAGCTTAT TAAGACTCCT 30 27 base pairs nucleicacid single linear DNA (genomic) 53 GATCCTTGTG TCCATATGAA ACAAAGC 27 39base pairs nucleic acid single linear DNA (genomic) 54 CACGTCGGTCGAGGATCCCT AACCACGGCC TTTAACCAG 39 74 base pairs nucleic acid singlelinear DNA (genomic) 55 TATGAAACAA AGCACTATTG CACTGGCACT CTTACCGTTACTGTTTACCC CGGTGACCAA 60 AGCCCACGCT GAAG 74 76 base pairs nucleic acidsingle linear DNA (genomic) 56 GTACCTTCAG CGTGGGCTTT GGTCACCGGGGTAAACAGTA ACGGTAAGAG TGCCAGTGCA 60 ATAGTGCTTT GTTTCA 76 27 base pairsnucleic acid single linear DNA (genomic) 57 CCGGACGCGT GGAGATTATCGTCACTG 27 29 base pairs nucleic acid single linear DNA (genomic) 58GCTTTGGTCA CCGGGGTAAA CAGTAACGG 29 42 base pairs nucleic acid singlelinear DNA (genomic) 59 CTGTTTACCC CGGTGACCAA AGCCGAGGTG TGCTCTGAAC AA42 36 base pairs nucleic acid single linear DNA (genomic) 60 AATAGCGGATCCGCACACTG CCATGCAGTA CTCTTC 36 35 base pairs nucleic acid single linearDNA (genomic) 61 GCTTTAAACC GGTAGGTGGC CCGGCTCCAT GCACC 35 36 base pairsnucleic acid single linear DNA (genomic) 62 CGAATTCACC GGTGTCATCCTCGGCACCGT CACCCT 36 42 base pairs nucleic acid single linear DNA(genomic) 63 GGGCTGAGAC CGGTCCGTGC CGTNCGCTGG TACTTTGACG TC 42 30 basepairs nucleic acid single linear DNA (genomic) 64 GGAATAGCGG ATCCGCACACTGCCATGCAG 30 4 amino acids amino acid single linear peptide 65 Ala AlaIle Phe 1 41 base pairs nucleic acid single linear DNA (genomic) 66GCTGAGACCG GTCCGTGCCG TNGCANTGGT ACTTTGACGT C 41 37 base pairs nucleicacid single linear DNA (genomic) 67 GCTGAGACCG GTCCGTGCCG TNTGGTACTTTGACGTC 37 52 base pairs nucleic acid single linear DNA (genomic) 68CTAGATAAAA GAGAGGTTGT TAGAGAGGTG TGCTCTGAAC AAGCTGAGGT TG 52 51 basepairs nucleic acid single linear DNA (genomic) 69 GACCAACCTC AGCTTGTTCAGAGCACACCT CTCTAACAAC CTCTCTTTTA T 51 92 base pairs nucleic acid singlelinear DNA (genomic) 70 CACTGAAGGT AAGTGCGCTC CATTCTTTTA CGGCGGTTGCTACGGCAACC GTAACAACTT 60 TGACACTGAA GAGTACTGCA TGGCAGTGTG CG 92 100 basepairs nucleic acid single linear DNA (genomic) 71 GATCCGCACA CTGCCATGCAGTACTCTTCA GTGTCAAAGT TGTTACGGTT GCCGTAGCAA 60 CCGCCGTAAA AGAATGGAGCGCACTTACCT TCAGTGACGT 100 92 base pairs nucleic acid single linear DNA(genomic) 72 CACTGAAGGT AAGTGCGCTC CATTCTTTTA CGGCGGTTGC TTGGGCAACCGTAACAACTT 60 TGACACTGAA GAGTACTGCA TGGCAGTGTG CG 92 100 base pairsnucleic acid single linear DNA (genomic) 73 GATCCGCACA CTGCCATGCAGTACTCTTCA GTGTCAAAGT TGTTACGGTT GCCCAAGCAA 60 CCGCCGTAAA AGAATGGAGCGCACTTACCT TCAGTGACGT 100 237 base pairs nucleic acid single linear DNA(genomic) CDS 2..235 74 T ATG AAA CAA AGC ACT ATT GCA CTG GCA CTC TTACCG TTA CTG TTT 46 Met Lys Gln Ser Thr Ile Ala Leu Ala Leu Leu Pro LeuLeu Phe 1 5 10 15 ACC CCT GTG ACA AAA GCC GAG GTG TGC TCT GAA CAA GCTGAG ACC GGT 94 Thr Pro Val Thr Lys Ala Glu Val Cys Ser Glu Gln Ala GluThr Gly 20 25 30 CCG TGC CGT GCA ATG ATC TCC CGC TGG TAC TTT GAC GTC ACTGAA GGT 142 Pro Cys Arg Ala Met Ile Ser Arg Trp Tyr Phe Asp Val Thr GluGly 35 40 45 AAG TGC GCT CCA TTC TTT TAC GGC GGT TGC GGC GGC AAC CGT AACAAC 190 Lys Cys Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn50 55 60 TTT GAC ACT GAA GAG TAC TGC ATG GCA GTG TGC GGA TCC GCT ATT 235Phe Asp Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 65 70 75 TA237 78 amino acids amino acid linear protein 75 Met Lys Gln Ser Thr IleAla Leu Ala Leu Leu Pro Leu Leu Phe Thr 1 5 10 15 Pro Val Thr Lys AlaGlu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro 20 25 30 Cys Arg Ala Met IleSer Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys 35 40 45 Cys Ala Pro Phe PheTyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe 50 55 60 Asp Thr Glu Glu TyrCys Met Ala Val Cys Gly Ser Ala Ile 65 70 75 185 base pairs nucleic acidsingle linear DNA (genomic) CDS 1..183 76 CTA GAT AAA AGA GAG GTG TGCTCT GAA CAA GCT GAG ACC GGT CCG TGC 48 Leu Asp Lys Arg Glu Val Cys SerGlu Gln Ala Glu Thr Gly Pro Cys 80 85 90 CGT GCA ATG ATC TCC CGC TGG TACTTT GAC GTC ACT GAA GGT AAG TGC 96 Arg Ala Met Ile Ser Arg Trp Tyr PheAsp Val Thr Glu Gly Lys Cys 95 100 105 110 GCT CCA TTC TTT TAC GGC GGTTGC GGC GGC AAC CGT AAC AAC TTT GAC 144 Ala Pro Phe Phe Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp 115 120 125 ACT GAA GAG TAC TGC ATG GCAGTG TGC GGA TCC GCT ATT TA 185 Thr Glu Glu Tyr Cys Met Ala Val Cys GlySer Ala Ile 130 135 61 amino acids amino acid linear protein 77 Leu AspLys Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 ArgAla Met Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 AlaPro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 ThrGlu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 197 base pairsnucleic acid single linear DNA (genomic) CDS 1..195 78 CTA GAT AAA AGAGAG GTT GTT AGA GAG GTG TGC TCT GAA CAA GCT GAG 48 Leu Asp Lys Arg GluVal Val Arg Glu Val Cys Ser Glu Gln Ala Glu 65 70 75 ACC GGT CCG TGC CGTGCA ATG ATC TCC CGC TGG TAC TTT GAC GTC ACT 96 Thr Gly Pro Cys Arg AlaMet Ile Ser Arg Trp Tyr Phe Asp Val Thr 80 85 90 GAA GGT AAG TGC GCT CCATTC TTT TAC GGC GGT TGC GGC GGC AAC CGT 144 Glu Gly Lys Cys Ala Pro PhePhe Tyr Gly Gly Cys Gly Gly Asn Arg 95 100 105 AAC AAC TTT GAC ACT GAAGAG TAC TGC ATG GCA GTG TGC GGA TCC GCT 192 Asn Asn Phe Asp Thr Glu GluTyr Cys Met Ala Val Cys Gly Ser Ala 110 115 120 125 ATT TA 197 Ile 65amino acids amino acid linear protein 79 Leu Asp Lys Arg Glu Val Val ArgGlu Val Cys Ser Glu Gln Ala Glu 1 5 10 15 Thr Gly Pro Cys Arg Ala MetIle Ser Arg Trp Tyr Phe Asp Val Thr 20 25 30 Glu Gly Lys Cys Ala Pro PhePhe Tyr Gly Gly Cys Gly Gly Asn Arg 35 40 45 Asn Asn Phe Asp Thr Glu GluTyr Cys Met Ala Val Cys Gly Ser Ala 50 55 60 Ile 65 445 base pairsnucleic acid single linear DNA (genomic) CDS 1..438 80 ATG AGA TTT CCTTCA ATT TTT ACT GCA GTT TTA TTC GCA GCA TCC TCC 48 Met Arg Phe Pro SerIle Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 70 75 80 GCA TTA GCT GCT CCAGTC AAC ACT ACA ACA GAA GAT GAA ACG GCA CAA 96 Ala Leu Ala Ala Pro ValAsn Thr Thr Thr Glu Asp Glu Thr Ala Gln 85 90 95 ATT CCG GCT GAA GCT GTCATC GGT TAC TTA GAT TTA GAA GGG GAT TTC 144 Ile Pro Ala Glu Ala Val IleGly Tyr Leu Asp Leu Glu Gly Asp Phe 100 105 110 GAT GTT GCT GTT TTG CCATTT TCC AAC AGC ACA AAT AAC GGG TTA TTG 192 Asp Val Ala Val Leu Pro PheSer Asn Ser Thr Asn Asn Gly Leu Leu 115 120 125 TTT ATA AAT ACT ACT ATTGCC AGC ATT GCT GCT AAA GAA GAA GGG GTA 240 Phe Ile Asn Thr Thr Ile AlaSer Ile Ala Ala Lys Glu Glu Gly Val 130 135 140 145 TCT CTA GAT AAA AGAGAG GTT GTT AGA GAG GTG TGC TCT GAA CAA GCT 288 Ser Leu Asp Lys Arg GluVal Val Arg Glu Val Cys Ser Glu Gln Ala 150 155 160 GAG ACC GGT CCG TGCCGT GCA ATG ATC TCC CGC TGG TAC TTT GAC GTC 336 Glu Thr Gly Pro Cys ArgAla Met Ile Ser Arg Trp Tyr Phe Asp Val 165 170 175 ACT GAA GGT AAG TGCGCT CCA TTC TTT TAC GGC GGT TGC GGC GGC AAC 384 Thr Glu Gly Lys Cys AlaPro Phe Phe Tyr Gly Gly Cys Gly Gly Asn 180 185 190 CGT AAC AAC TTT GACACT GAA GAG TAC TGC ATG GCA GTG TGC GGA TCC 432 Arg Asn Asn Phe Asp ThrGlu Glu Tyr Cys Met Ala Val Cys Gly Ser 195 200 205 GCT ATT TAAGCTT 445Ala Ile 210 146 amino acids amino acid linear protein 81 Met Arg Phe ProSer Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu AlaAla Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln 20 25 30 Ile Pro AlaGlu Ala Val Ile Gly Tyr Leu Asp Leu Glu Gly Asp Phe 35 40 45 Asp Val AlaVal Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu 50 55 60 Phe Ile AsnThr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val 65 70 75 80 Ser LeuAsp Lys Arg Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala 85 90 95 Glu ThrGly Pro Cys Arg Ala Met Ile Ser Arg Trp Tyr Phe Asp Val 100 105 110 ThrGlu Gly Lys Cys Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn 115 120 125Arg Asn Asn Phe Asp Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser 130 135140 Ala Ile 145 445 base pairs nucleic acid single linear DNA (genomic)CDS 1..438 82 ATG AGA TTT CCT TCA ATT TTT ACT GCA GTT TTA TTC GCA GCATCC TCC 48 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala SerSer 150 155 160 GCA TTA GCT GCT CCA GTC AAC ACT ACA ACA GAA GAT GAA ACGGCA CAA 96 Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr AlaGln 165 170 175 ATT CCG GCT GAA GCT GTC ATC GGT TAC TTA GAT TTA GAA GGGGAT TTC 144 Ile Pro Ala Glu Ala Val Ile Gly Tyr Leu Asp Leu Glu Gly AspPhe 180 185 190 GAT GTT GCT GTT TTG CCA TTT TCC AAC AGC ACA AAT AAC GGGTTA TTG 192 Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly LeuLeu 195 200 205 210 TTT ATA AAT ACT ACT ATT GCC AGC ATT GCT GCT AAA GAAGAA GGG GTA 240 Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu GluGly Val 215 220 225 TCT CTA GAT AAA AGA GAG GTT GTT AGA GAG GTG TGC TCTGAA CAA GCT 288 Ser Leu Asp Lys Arg Glu Val Val Arg Glu Val Cys Ser GluGln Ala 230 235 240 GAG ACC GGT CCG TGC CGT GCA GCT ATC TGG CGC TGG TACTTT GAC GTC 336 Glu Thr Gly Pro Cys Arg Ala Ala Ile Trp Arg Trp Tyr PheAsp Val 245 250 255 ACT GAA GGT AAG TGC GCT CCA TTC TTT TAC GGC GGT TGCGGC GGC AAC 384 Thr Glu Gly Lys Cys Ala Pro Phe Phe Tyr Gly Gly Cys GlyGly Asn 260 265 270 CGT AAC AAC TTT GAC ACT GAA GAG TAC TGC ATG GCA GTGTGC GGA TCC 432 Arg Asn Asn Phe Asp Thr Glu Glu Tyr Cys Met Ala Val CysGly Ser 275 280 285 290 GCT ATT TAAGCTT 445 Ala Ile 146 amino acidsamino acid linear protein 83 Met Arg Phe Pro Ser Ile Phe Thr Ala Val LeuPhe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala Ala Pro Val Asn Thr Thr ThrGlu Asp Glu Thr Ala Gln 20 25 30 Ile Pro Ala Glu Ala Val Ile Gly Tyr LeuAsp Leu Glu Gly Asp Phe 35 40 45 Asp Val Ala Val Leu Pro Phe Ser Asn SerThr Asn Asn Gly Leu Leu 50 55 60 Phe Ile Asn Thr Thr Ile Ala Ser Ile AlaAla Lys Glu Glu Gly Val 65 70 75 80 Ser Leu Asp Lys Arg Glu Val Val ArgGlu Val Cys Ser Glu Gln Ala 85 90 95 Glu Thr Gly Pro Cys Arg Ala Ala IleTrp Arg Trp Tyr Phe Asp Val 100 105 110 Thr Glu Gly Lys Cys Ala Pro PhePhe Tyr Gly Gly Cys Gly Gly Asn 115 120 125 Arg Asn Asn Phe Asp Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser 130 135 140 Ala Ile 145 445 basepairs nucleic acid single linear DNA (genomic) CDS 1..438 84 ATG AGA TTTCCT TCA ATT TTT ACT GCA GTT TTA TTC GCA GCA TCC TCC 48 Met Arg Phe ProSer Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 150 155 160 GCA TTA GCTGCT CCA GTC AAC ACT ACA ACA GAA GAT GAA ACG GCA CAA 96 Ala Leu Ala AlaPro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln 165 170 175 ATT CCG GCTGAA GCT GTC ATC GGT TAC TTA GAT TTA GAA GGG GAT TTC 144 Ile Pro Ala GluAla Val Ile Gly Tyr Leu Asp Leu Glu Gly Asp Phe 180 185 190 GAT GTT GCTGTT TTG CCA TTT TCC AAC AGC ACA AAT AAC GGG TTA TTG 192 Asp Val Ala ValLeu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu 195 200 205 210 TTT ATAAAT ACT ACT ATT GCC AGC ATT GCT GCT AAA GAA GAA GGG GTA 240 Phe Ile AsnThr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val 215 220 225 TCT CTAGAT AAA AGA GAG GTT GTT AGA GAG GTG TGC TCT GAA CAA GCT 288 Ser Leu AspLys Arg Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala 230 235 240 GAG ACCGGT CCG TGC CGT GCA GCT ATC TAC CGC TGG TAC TTT GAC GTC 336 Glu Thr GlyPro Cys Arg Ala Ala Ile Tyr Arg Trp Tyr Phe Asp Val 245 250 255 ACT GAAGGT AAG TGC GCT CCA TTC TTT TAC GGC GGT TGC GGC GGC AAC 384 Thr Glu GlyLys Cys Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn 260 265 270 CGT AACAAC TTT GAC ACT GAA GAG TAC TGC ATG GCA GTG TGC GGA TCC 432 Arg Asn AsnPhe Asp Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser 275 280 285 290 GCTATT TAAGCTT 445 Ala Ile 146 amino acids amino acid linear protein 85 MetArg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln 20 25 30Ile Pro Ala Glu Ala Val Ile Gly Tyr Leu Asp Leu Glu Gly Asp Phe 35 40 45Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu 50 55 60Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val 65 70 7580 Ser Leu Asp Lys Arg Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala 85 9095 Glu Thr Gly Pro Cys Arg Ala Ala Ile Tyr Arg Trp Tyr Phe Asp Val 100105 110 Thr Glu Gly Lys Cys Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn115 120 125 Arg Asn Asn Phe Asp Thr Glu Glu Tyr Cys Met Ala Val Cys GlySer 130 135 140 Ala Ile 145 445 base pairs nucleic acid single linearDNA (genomic) CDS 1..438 86 ATG AGA TTT CCT TCA ATT TTT ACT GCA GTT TTATTC GCA GCA TCC TCC 48 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu PheAla Ala Ser Ser 150 155 160 GCA TTA GCT GCT CCA GTC AAC ACT ACA ACA GAAGAT GAA ACG GCA CAA 96 Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu AspGlu Thr Ala Gln 165 170 175 ATT CCG GCT GAA GCT GTC ATC GGT TAC TTA GATTTA GAA GGG GAT TTC 144 Ile Pro Ala Glu Ala Val Ile Gly Tyr Leu Asp LeuGlu Gly Asp Phe 180 185 190 GAT GTT GCT GTT TTG CCA TTT TCC AAC AGC ACAAAT AAC GGG TTA TTG 192 Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr AsnAsn Gly Leu Leu 195 200 205 210 TTT ATA AAT ACT ACT ATT GCC AGC ATT GCTGCT AAA GAA GAA GGG GTA 240 Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala AlaLys Glu Glu Gly Val 215 220 225 TCT CTA GAT AAA AGA GAG GTT GTT AGA GAGGTG TGC TCT GAA CAA GCT 288 Ser Leu Asp Lys Arg Glu Val Val Arg Glu ValCys Ser Glu Gln Ala 230 235 240 GAG ACC GGT CCG TGC CGT GCA TTG ATC TTCCGC TGG TAC TTT GAC GTC 336 Glu Thr Gly Pro Cys Arg Ala Leu Ile Phe ArgTrp Tyr Phe Asp Val 245 250 255 ACT GAA GGT AAG TGC GCT CCA TTC TTT TACGGC GGT TGC GGC GGC AAC 384 Thr Glu Gly Lys Cys Ala Pro Phe Phe Tyr GlyGly Cys Gly Gly Asn 260 265 270 CGT AAC AAC TTT GAC ACT GAA GAG TAC TGCATG GCA GTG TGC GGA TCC 432 Arg Asn Asn Phe Asp Thr Glu Glu Tyr Cys MetAla Val Cys Gly Ser 275 280 285 290 GCT ATT TAAGCTT 445 Ala Ile 146amino acids amino acid linear protein 87 Met Arg Phe Pro Ser Ile Phe ThrAla Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala Ala Pro Val AsnThr Thr Thr Glu Asp Glu Thr Ala Gln 20 25 30 Ile Pro Ala Glu Ala Val IleGly Tyr Leu Asp Leu Glu Gly Asp Phe 35 40 45 Asp Val Ala Val Leu Pro PheSer Asn Ser Thr Asn Asn Gly Leu Leu 50 55 60 Phe Ile Asn Thr Thr Ile AlaSer Ile Ala Ala Lys Glu Glu Gly Val 65 70 75 80 Ser Leu Asp Lys Arg GluVal Val Arg Glu Val Cys Ser Glu Gln Ala 85 90 95 Glu Thr Gly Pro Cys ArgAla Leu Ile Phe Arg Trp Tyr Phe Asp Val 100 105 110 Thr Glu Gly Lys CysAla Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn 115 120 125 Arg Asn Asn PheAsp Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser 130 135 140 Ala Ile 145445 base pairs nucleic acid single linear DNA (genomic) CDS 1..438 88ATG AGA TTT CCT TCA ATT TTT ACT GCA GTT TTA TTC GCA GCA TCC TCC 48 MetArg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 150 155 160GCA TTA GCT GCT CCA GTC AAC ACT ACA ACA GAA GAT GAA ACG GCA CAA 96 AlaLeu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln 165 170 175ATT CCG GCT GAA GCT GTC ATC GGT TAC TTA GAT TTA GAA GGG GAT TTC 144 IlePro Ala Glu Ala Val Ile Gly Tyr Leu Asp Leu Glu Gly Asp Phe 180 185 190GAT GTT GCT GTT TTG CCA TTT TCC AAC AGC ACA AAT AAC GGG TTA TTG 192 AspVal Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu 195 200 205210 TTT ATA AAT ACT ACT ATT GCC AGC ATT GCT GCT AAA GAA GAA GGG GTA 240Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val 215 220225 TCT CTA GAT AAA AGA GAG GTT GTT AGA GAG GTG TGC TCT GAA CAA GCT 288Ser Leu Asp Lys Arg Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala 230 235240 GAG ACC GGT CCG TGC CGT GCA TTG ATC TAC CGC TGG TAC TTT GAC GTC 336Glu Thr Gly Pro Cys Arg Ala Leu Ile Tyr Arg Trp Tyr Phe Asp Val 245 250255 ACT GAA GGT AAG TGC GCT CCA TTC TTT TAC GGC GGT TGC GGC GGC AAC 384Thr Glu Gly Lys Cys Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn 260 265270 CGT AAC AAC TTT GAC ACT GAA GAG TAC TGC ATG GCA GTG TGC GGA TCC 432Arg Asn Asn Phe Asp Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser 275 280285 290 GCT ATT TAAGCTT 445 Ala Ile 146 amino acids amino acid linearprotein 89 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala SerSer 1 5 10 15 Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu ThrAla Gln 20 25 30 Ile Pro Ala Glu Ala Val Ile Gly Tyr Leu Asp Leu Glu GlyAsp Phe 35 40 45 Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn GlyLeu Leu 50 55 60 Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu GluGly Val 65 70 75 80 Ser Leu Asp Lys Arg Glu Val Val Arg Glu Val Cys SerGlu Gln Ala 85 90 95 Glu Thr Gly Pro Cys Arg Ala Leu Ile Tyr Arg Trp TyrPhe Asp Val 100 105 110 Thr Glu Gly Lys Cys Ala Pro Phe Phe Tyr Gly GlyCys Gly Gly Asn 115 120 125 Arg Asn Asn Phe Asp Thr Glu Glu Tyr Cys MetAla Val Cys Gly Ser 130 135 140 Ala Ile 145 445 base pairs nucleic acidsingle linear DNA (genomic) CDS 1..438 90 ATG AGA TTT CCT TCA ATT TTTACT GCA GTT TTA TTC GCA GCA TCC TCC 48 Met Arg Phe Pro Ser Ile Phe ThrAla Val Leu Phe Ala Ala Ser Ser 150 155 160 GCA TTA GCT GCT CCA GTC AACACT ACA ACA GAA GAT GAA ACG GCA CAA 96 Ala Leu Ala Ala Pro Val Asn ThrThr Thr Glu Asp Glu Thr Ala Gln 165 170 175 ATT CCG GCT GAA GCT GTC ATCGGT TAC TTA GAT TTA GAA GGG GAT TTC 144 Ile Pro Ala Glu Ala Val Ile GlyTyr Leu Asp Leu Glu Gly Asp Phe 180 185 190 GAT GTT GCT GTT TTG CCA TTTTCC AAC AGC ACA AAT AAC GGG TTA TTG 192 Asp Val Ala Val Leu Pro Phe SerAsn Ser Thr Asn Asn Gly Leu Leu 195 200 205 210 TTT ATA AAT ACT ACT ATTGCC AGC ATT GCT GCT AAA GAA GAA GGG GTA 240 Phe Ile Asn Thr Thr Ile AlaSer Ile Ala Ala Lys Glu Glu Gly Val 215 220 225 TCT CTA GAT AAA AGA GAGGTT GTT AGA GAG GTG TGC TCT GAA CAA GCT 288 Ser Leu Asp Lys Arg Glu ValVal Arg Glu Val Cys Ser Glu Gln Ala 230 235 240 GAG ACC GGT CCG TGC CGTGCA ATG CAC TTC CGC TGG TAC TTT GAC GTC 336 Glu Thr Gly Pro Cys Arg AlaMet His Phe Arg Trp Tyr Phe Asp Val 245 250 255 ACT GAA GGT AAG TGC GCTCCA TTC TTT TAC GGC GGT TGC GGC GGC AAC 384 Thr Glu Gly Lys Cys Ala ProPhe Phe Tyr Gly Gly Cys Gly Gly Asn 260 265 270 CGT AAC AAC TTT GAC ACTGAA GAG TAC TGC ATG GCA GTG TGC GGA TCC 432 Arg Asn Asn Phe Asp Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser 275 280 285 290 GCT ATT TAAGCTT 445Ala Ile 146 amino acids amino acid linear protein 91 Met Arg Phe Pro SerIle Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala AlaPro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln 20 25 30 Ile Pro Ala GluAla Val Ile Gly Tyr Leu Asp Leu Glu Gly Asp Phe 35 40 45 Asp Val Ala ValLeu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu 50 55 60 Phe Ile Asn ThrThr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val 65 70 75 80 Ser Leu AspLys Arg Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala 85 90 95 Glu Thr GlyPro Cys Arg Ala Met His Phe Arg Trp Tyr Phe Asp Val 100 105 110 Thr GluGly Lys Cys Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn 115 120 125 ArgAsn Asn Phe Asp Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser 130 135 140Ala Ile 145 445 base pairs nucleic acid single linear DNA (genomic) CDS1..438 92 ATG AGA TTT CCT TCA ATT TTT ACT GCA GTT TTA TTC GCA GCA TCCTCC 48 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser150 155 160 GCA TTA GCT GCT CCA GTC AAC ACT ACA ACA GAA GAT GAA ACG GCACAA 96 Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln165 170 175 ATT CCG GCT GAA GCT GTC ATC GGT TAC TTA GAT TTA GAA GGG GATTTC 144 Ile Pro Ala Glu Ala Val Ile Gly Tyr Leu Asp Leu Glu Gly Asp Phe180 185 190 GAT GTT GCT GTT TTG CCA TTT TCC AAC AGC ACA AAT AAC GGG TTATTG 192 Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu195 200 205 210 TTT ATA AAT ACT ACT ATT GCC AGC ATT GCT GCT AAA GAA GAAGGG GTA 240 Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu GlyVal 215 220 225 TCT CTA GAT AAA AGA GAG GTT GTT AGA GAG GTG TGC TCT GAACAA GCT 288 Ser Leu Asp Lys Arg Glu Val Val Arg Glu Val Cys Ser Glu GlnAla 230 235 240 GAG ACC GGT CCG TGC CGT GCA ATG CAC TAC CGC TGG TAC TTTGAC GTC 336 Glu Thr Gly Pro Cys Arg Ala Met His Tyr Arg Trp Tyr Phe AspVal 245 250 255 ACT GAA GGT AAG TGC GCT CCA TTC TTT TAC GGC GGT TGC GGCGGC AAC 384 Thr Glu Gly Lys Cys Ala Pro Phe Phe Tyr Gly Gly Cys Gly GlyAsn 260 265 270 CGT AAC AAC TTT GAC ACT GAA GAG TAC TGC ATG GCA GTG TGCGGA TCC 432 Arg Asn Asn Phe Asp Thr Glu Glu Tyr Cys Met Ala Val Cys GlySer 275 280 285 290 GCT ATT TAAGCTT 445 Ala Ile 146 amino acids aminoacid linear protein 93 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu PheAla Ala Ser Ser 1 5 10 15 Ala Leu Ala Ala Pro Val Asn Thr Thr Thr GluAsp Glu Thr Ala Gln 20 25 30 Ile Pro Ala Glu Ala Val Ile Gly Tyr Leu AspLeu Glu Gly Asp Phe 35 40 45 Asp Val Ala Val Leu Pro Phe Ser Asn Ser ThrAsn Asn Gly Leu Leu 50 55 60 Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala AlaLys Glu Glu Gly Val 65 70 75 80 Ser Leu Asp Lys Arg Glu Val Val Arg GluVal Cys Ser Glu Gln Ala 85 90 95 Glu Thr Gly Pro Cys Arg Ala Met His TyrArg Trp Tyr Phe Asp Val 100 105 110 Thr Glu Gly Lys Cys Ala Pro Phe PheTyr Gly Gly Cys Gly Gly Asn 115 120 125 Arg Asn Asn Phe Asp Thr Glu GluTyr Cys Met Ala Val Cys Gly Ser 130 135 140 Ala Ile 145 445 base pairsnucleic acid single linear DNA (genomic) CDS 1..438 94 ATG AGA TTT CCTTCA ATT TTT ACT GCA GTT TTA TTC GCA GCA TCC TCC 48 Met Arg Phe Pro SerIle Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 150 155 160 GCA TTA GCT GCTCCA GTC AAC ACT ACA ACA GAA GAT GAA ACG GCA CAA 96 Ala Leu Ala Ala ProVal Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln 165 170 175 ATT CCG GCT GAAGCT GTC ATC GGT TAC TTA GAT TTA GAA GGG GAT TTC 144 Ile Pro Ala Glu AlaVal Ile Gly Tyr Leu Asp Leu Glu Gly Asp Phe 180 185 190 GAT GTT GCT GTTTTG CCA TTT TCC AAC AGC ACA AAT AAC GGG TTA TTG 192 Asp Val Ala Val LeuPro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu 195 200 205 210 TTT ATA AATACT ACT ATT GCC AGC ATT GCT GCT AAA GAA GAA GGG GTA 240 Phe Ile Asn ThrThr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val 215 220 225 TCT CTA GATAAA AGA GAG GTT GTT AGA GAG GTG TGC TCT GAA CAA GCT 288 Ser Leu Asp LysArg Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala 230 235 240 GAG ACC GGTCCG TGC CGT GCA ATG CAC TGG CGC TGG TAC TTT GAC GTC 336 Glu Thr Gly ProCys Arg Ala Met His Trp Arg Trp Tyr Phe Asp Val 245 250 255 ACT GAA GGTAAG TGC GCT CCA TTC TTT TAC GGC GGT TGC GGC GGC AAC 384 Thr Glu Gly LysCys Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn 260 265 270 CGT AAC AACTTT GAC ACT GAA GAG TAC TGC ATG GCA GTG TGC GGA TCC 432 Arg Asn Asn PheAsp Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser 275 280 285 290 GCT ATTTAAGCTT 445 Ala Ile 146 amino acids amino acid linear protein 95 Met ArgPhe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 AlaLeu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln 20 25 30 IlePro Ala Glu Ala Val Ile Gly Tyr Leu Asp Leu Glu Gly Asp Phe 35 40 45 AspVal Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu 50 55 60 PheIle Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val 65 70 75 80Ser Leu Asp Lys Arg Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala 85 90 95Glu Thr Gly Pro Cys Arg Ala Met His Trp Arg Trp Tyr Phe Asp Val 100 105110 Thr Glu Gly Lys Cys Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn 115120 125 Arg Asn Asn Phe Asp Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser130 135 140 Ala Ile 145 445 base pairs nucleic acid single linear DNA(genomic) CDS 1..438 96 ATG AGA TTT CCT TCA ATT TTT ACT GCA GTT TTA TTCGCA GCA TCC TCC 48 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe AlaAla Ser Ser 150 155 160 GCA TTA GCT GCT CCA GTC AAC ACT ACA ACA GAA GATGAA ACG GCA CAA 96 Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp GluThr Ala Gln 165 170 175 ATT CCG GCT GAA GCT GTC ATC GGT TAC TTA GAT TTAGAA GGG GAT TTC 144 Ile Pro Ala Glu Ala Val Ile Gly Tyr Leu Asp Leu GluGly Asp Phe 180 185 190 GAT GTT GCT GTT TTG CCA TTT TCC AAC AGC ACA AATAAC GGG TTA TTG 192 Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn AsnGly Leu Leu 195 200 205 210 TTT ATA AAT ACT ACT ATT GCC AGC ATT GCT GCTAAA GAA GAA GGG GTA 240 Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala LysGlu Glu Gly Val 215 220 225 TCT CTA GAT AAA AGA GAG GTT GTT AGA GAG GTGTGC TCT GAA CAA GCT 288 Ser Leu Asp Lys Arg Glu Val Val Arg Glu Val CysSer Glu Gln Ala 230 235 240 GAG ACC GGT CCG TGC CGT GCA GCT CAC TCC CGCTGG TAC TTT GAC GTC 336 Glu Thr Gly Pro Cys Arg Ala Ala His Ser Arg TrpTyr Phe Asp Val 245 250 255 ACT GAA GGT AAG TGC GCT CCA TTC TTT TAC GGCGGT TGC GGC GGC AAC 384 Thr Glu Gly Lys Cys Ala Pro Phe Phe Tyr Gly GlyCys Gly Gly Asn 260 265 270 CGT AAC AAC TTT GAC ACT GAA GAG TAC TGC ATGGCA GTG TGC GGA TCC 432 Arg Asn Asn Phe Asp Thr Glu Glu Tyr Cys Met AlaVal Cys Gly Ser 275 280 285 290 GCT ATT TAAGCTT 445 Ala Ile 146 aminoacids amino acid linear protein 97 Met Arg Phe Pro Ser Ile Phe Thr AlaVal Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala Ala Pro Val Asn ThrThr Thr Glu Asp Glu Thr Ala Gln 20 25 30 Ile Pro Ala Glu Ala Val Ile GlyTyr Leu Asp Leu Glu Gly Asp Phe 35 40 45 Asp Val Ala Val Leu Pro Phe SerAsn Ser Thr Asn Asn Gly Leu Leu 50 55 60 Phe Ile Asn Thr Thr Ile Ala SerIle Ala Ala Lys Glu Glu Gly Val 65 70 75 80 Ser Leu Asp Lys Arg Glu ValVal Arg Glu Val Cys Ser Glu Gln Ala 85 90 95 Glu Thr Gly Pro Cys Arg AlaAla His Ser Arg Trp Tyr Phe Asp Val 100 105 110 Thr Glu Gly Lys Cys AlaPro Phe Phe Tyr Gly Gly Cys Gly Gly Asn 115 120 125 Arg Asn Asn Phe AspThr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser 130 135 140 Ala Ile 145 445base pairs nucleic acid single linear DNA (genomic) CDS 1..438 98 ATGAGA TTT CCT TCA ATT TTT ACT GCA GTT TTA TTC GCA GCA TCC TCC 48 Met ArgPhe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 150 155 160 GCATTA GCT GCT CCA GTC AAC ACT ACA ACA GAA GAT GAA ACG GCA CAA 96 Ala LeuAla Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln 165 170 175 ATTCCG GCT GAA GCT GTC ATC GGT TAC TTA GAT TTA GAA GGG GAT TTC 144 Ile ProAla Glu Ala Val Ile Gly Tyr Leu Asp Leu Glu Gly Asp Phe 180 185 190 GATGTT GCT GTT TTG CCA TTT TCC AAC AGC ACA AAT AAC GGG TTA TTG 192 Asp ValAla Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu 195 200 205 210TTT ATA AAT ACT ACT ATT GCC AGC ATT GCT GCT AAA GAA GAA GGG GTA 240 PheIle Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val 215 220 225TCT CTA GAT AAA AGA GAG GTT GTT AGA GAG GTG TGC TCT GAA CAA GCT 288 SerLeu Asp Lys Arg Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala 230 235 240GAG ACC GGT CCG TGC CGT GCA TTG CAC TCC CGC TGG TAC TTT GAC GTC 336 GluThr Gly Pro Cys Arg Ala Leu His Ser Arg Trp Tyr Phe Asp Val 245 250 255ACT GAA GGT AAG TGC GCT CCA TTC TTT TAC GGC GGT TGC GGC GGC AAC 384 ThrGlu Gly Lys Cys Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn 260 265 270CGT AAC AAC TTT GAC ACT GAA GAG TAC TGC ATG GCA GTG TGC GGA TCC 432 ArgAsn Asn Phe Asp Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser 275 280 285290 GCT ATT TAAGCTT 445 Ala Ile 146 amino acids amino acid linearprotein 99 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala SerSer 1 5 10 15 Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu ThrAla Gln 20 25 30 Ile Pro Ala Glu Ala Val Ile Gly Tyr Leu Asp Leu Glu GlyAsp Phe 35 40 45 Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn GlyLeu Leu 50 55 60 Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu GluGly Val 65 70 75 80 Ser Leu Asp Lys Arg Glu Val Val Arg Glu Val Cys SerGlu Gln Ala 85 90 95 Glu Thr Gly Pro Cys Arg Ala Leu His Ser Arg Trp TyrPhe Asp Val 100 105 110 Thr Glu Gly Lys Cys Ala Pro Phe Phe Tyr Gly GlyCys Gly Gly Asn 115 120 125 Arg Asn Asn Phe Asp Thr Glu Glu Tyr Cys MetAla Val Cys Gly Ser 130 135 140 Ala Ile 145 704 base pairs nucleic acidsingle linear DNA (genomic) CDS 1..699 100 GTG AAA CAA AGC ACT ATT GCACTG GCA CTC TTA CCG TTA CTG TTT ACC 48 Val Lys Gln Ser Thr Ile Ala LeuAla Leu Leu Pro Leu Leu Phe Thr 150 155 160 CCG GTG ACC AAA GCC GAG GTGTGC TCT GAA CAA GCT GAG ACC GGT CCG 96 Pro Val Thr Lys Ala Glu Val CysSer Glu Gln Ala Glu Thr Gly Pro 165 170 175 TGC CGT GCA ATG ATC TCC CGCTGG TAC TTT GAC GTC ACT GAA GGT AAG 144 Cys Arg Ala Met Ile Ser Arg TrpTyr Phe Asp Val Thr Glu Gly Lys 180 185 190 TGC GCT CCA TTC TTT TAC GGCGGT TGC GGC GGC AAC CGT AAC AAC TTT 192 Cys Ala Pro Phe Phe Tyr Gly GlyCys Gly Gly Asn Arg Asn Asn Phe 195 200 205 210 GAC ACT GAA GAG TAC TGCATG GCA GTG TGC GGA TCC GGT GGT GGC TCT 240 Asp Thr Glu Glu Tyr Cys MetAla Val Cys Gly Ser Gly Gly Gly Ser 215 220 225 GGT TCC GGT GAT TTT GATTAT GAA AAG ATG GCA AAC GCT AAT AAG GGG 288 Gly Ser Gly Asp Phe Asp TyrGlu Lys Met Ala Asn Ala Asn Lys Gly 230 235 240 GCT ATG ACC GAA AAT GCCGAT GAA AAC GCG CTA CAG TCT GAC GCT AAA 336 Ala Met Thr Glu Asn Ala AspGlu Asn Ala Leu Gln Ser Asp Ala Lys 245 250 255 GGC AAA CTT GAT TCT GTCGCT ACT GAT TAC GGT GCT GCT ATC GAT GGT 384 Gly Lys Leu Asp Ser Val AlaThr Asp Tyr Gly Ala Ala Ile Asp Gly 260 265 270 TTC ATT GGT GAC GTT TCCGGC CTT GCT AAT GGT AAT GGT GCT ACT GGT 432 Phe Ile Gly Asp Val Ser GlyLeu Ala Asn Gly Asn Gly Ala Thr Gly 275 280 285 290 GAT TTT GCT GGC TCTAAT TCC CAA ATG GCT CAA GTC GGT GAC GGT GAT 480 Asp Phe Ala Gly Ser AsnSer Gln Met Ala Gln Val Gly Asp Gly Asp 295 300 305 AAT TCA CCT TTA ATGAAT AAT TTC CGT CAA TAT TTA CCT TCC CTC CCT 528 Asn Ser Pro Leu Met AsnAsn Phe Arg Gln Tyr Leu Pro Ser Leu Pro 310 315 320 CAA TCG GTT GAA TGTCGC CCT TTT GTC TTT GGC GCT GGT AAA CCA TAC 576 Gln Ser Val Glu Cys ArgPro Phe Val Phe Gly Ala Gly Lys Pro Tyr 325 330 335 GAA TTT TCT ATT GATTGT GAC AAA ATA AAC TTA TTC CGT GGT GTC TTT 624 Glu Phe Ser Ile Asp CysAsp Lys Ile Asn Leu Phe Arg Gly Val Phe 340 345 350 GCG TTT CTT TTA TATGTT GCC ACC TTT ATG TAT GTA TTT TCT ACG TTT 672 Ala Phe Leu Leu Tyr ValAla Thr Phe Met Tyr Val Phe Ser Thr Phe 355 360 365 370 GCT AAC ATA CTGCGT AAT AAG GAG TCT TAATA 704 Ala Asn Ile Leu Arg Asn Lys Glu Ser 375233 amino acids amino acid linear protein 101 Val Lys Gln Ser Thr IleAla Leu Ala Leu Leu Pro Leu Leu Phe Thr 1 5 10 15 Pro Val Thr Lys AlaGlu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro 20 25 30 Cys Arg Ala Met IleSer Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys 35 40 45 Cys Ala Pro Phe PheTyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe 50 55 60 Asp Thr Glu Glu TyrCys Met Ala Val Cys Gly Ser Gly Gly Gly Ser 65 70 75 80 Gly Ser Gly AspPhe Asp Tyr Glu Lys Met Ala Asn Ala Asn Lys Gly 85 90 95 Ala Met Thr GluAsn Ala Asp Glu Asn Ala Leu Gln Ser Asp Ala Lys 100 105 110 Gly Lys LeuAsp Ser Val Ala Thr Asp Tyr Gly Ala Ala Ile Asp Gly 115 120 125 Phe IleGly Asp Val Ser Gly Leu Ala Asn Gly Asn Gly Ala Thr Gly 130 135 140 AspPhe Ala Gly Ser Asn Ser Gln Met Ala Gln Val Gly Asp Gly Asp 145 150 155160 Asn Ser Pro Leu Met Asn Asn Phe Arg Gln Tyr Leu Pro Ser Leu Pro 165170 175 Gln Ser Val Glu Cys Arg Pro Phe Val Phe Gly Ala Gly Lys Pro Tyr180 185 190 Glu Phe Ser Ile Asp Cys Asp Lys Ile Asn Leu Phe Arg Gly ValPhe 195 200 205 Ala Phe Leu Leu Tyr Val Ala Thr Phe Met Tyr Val Phe SerThr Phe 210 215 220 Ala Asn Ile Leu Arg Asn Lys Glu Ser 225 230 701 basepairs nucleic acid single linear DNA (genomic) CDS 1..696 102 GTG AAACAA AGC ACT ATT GCA CTG GCA CTC TTA CCG TTA CTG TTT ACC 48 Val Lys GlnSer Thr Ile Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr 235 240 245 CCG GTGACC AAA GCC GAG GTG TGC TCT GAA CAA GCT GAG ACC GGT CCG 96 Pro Val ThrLys Ala Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro 250 255 260 265 TGCCGT NNS NNS NNS NNS TGG TAC TTT GAC GTC ACT GAA GGT AAG TGC 144 Cys ArgXaa Xaa Xaa Xaa Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 270 275 280 GCTCCA TTC TTT TAC GGC GGT TGC GGC GGC AAC CGT AAC AAC TTT GAC 192 Ala ProPhe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 285 290 295 ACTGAA GAG TAC TGC ATG GCA GTG TGC GGA TCC GGT GGT GGC TCT GGT 240 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Gly Gly Gly Ser Gly 300 305 310 TCCGGT GAT TTT GAT TAT GAA AAG ATG GCA AAC GCT AAT AAG GGG GCT 288 Ser GlyAsp Phe Asp Tyr Glu Lys Met Ala Asn Ala Asn Lys Gly Ala 315 320 325 ATGACC GAA AAT GCC GAT GAA AAC GCG CTA CAG TCT GAC GCT AAA GGC 336 Met ThrGlu Asn Ala Asp Glu Asn Ala Leu Gln Ser Asp Ala Lys Gly 330 335 340 345AAA CTT GAT TCT GTC GCT ACT GAT TAC GGT GCT GCT ATC GAT GGT TTC 384 LysLeu Asp Ser Val Ala Thr Asp Tyr Gly Ala Ala Ile Asp Gly Phe 350 355 360ATT GGT GAC GTT TCC GGC CTT GCT AAT GGT AAT GGT GCT ACT GGT GAT 432 IleGly Asp Val Ser Gly Leu Ala Asn Gly Asn Gly Ala Thr Gly Asp 365 370 375TTT GCT GGC TCT AAT TCC CAA ATG GCT CAA GTC GGT GAC GGT GAT AAT 480 PheAla Gly Ser Asn Ser Gln Met Ala Gln Val Gly Asp Gly Asp Asn 380 385 390TCA CCT TTA ATG AAT AAT TTC CGT CAA TAT TTA CCT TCC CTC CCT CAA 528 SerPro Leu Met Asn Asn Phe Arg Gln Tyr Leu Pro Ser Leu Pro Gln 395 400 405TCG GTT GAA TGT CGC CCT TTT GTC TTT GGC GCT GGT AAA CCA TAC GAA 576 SerVal Glu Cys Arg Pro Phe Val Phe Gly Ala Gly Lys Pro Tyr Glu 410 415 420425 TTT TCT ATT GAT TGT GAC AAA ATA AAC TTA TTC CGT GGT GTC TTT GCG 624Phe Ser Ile Asp Cys Asp Lys Ile Asn Leu Phe Arg Gly Val Phe Ala 430 435440 TTT CTT TTA TAT GTT GCC ACC TTT ATG TAT GTA TTT TCT ACG TTT GCT 672Phe Leu Leu Tyr Val Ala Thr Phe Met Tyr Val Phe Ser Thr Phe Ala 445 450455 AAC ATA CTG CGT AAT AAG GAG TCT TAATA 701 Asn Ile Leu Arg Asn LysGlu Ser 460 465 232 amino acids amino acid linear protein 103 Val LysGln Ser Thr Ile Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr 1 5 10 15 ProVal Thr Lys Ala Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro 20 25 30 CysArg Xaa Xaa Xaa Xaa Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 35 40 45 AlaPro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 50 55 60 ThrGlu Glu Tyr Cys Met Ala Val Cys Gly Ser Gly Gly Gly Ser Gly 65 70 75 80Ser Gly Asp Phe Asp Tyr Glu Lys Met Ala Asn Ala Asn Lys Gly Ala 85 90 95Met Thr Glu Asn Ala Asp Glu Asn Ala Leu Gln Ser Asp Ala Lys Gly 100 105110 Lys Leu Asp Ser Val Ala Thr Asp Tyr Gly Ala Ala Ile Asp Gly Phe 115120 125 Ile Gly Asp Val Ser Gly Leu Ala Asn Gly Asn Gly Ala Thr Gly Asp130 135 140 Phe Ala Gly Ser Asn Ser Gln Met Ala Gln Val Gly Asp Gly AspAsn 145 150 155 160 Ser Pro Leu Met Asn Asn Phe Arg Gln Tyr Leu Pro SerLeu Pro Gln 165 170 175 Ser Val Glu Cys Arg Pro Phe Val Phe Gly Ala GlyLys Pro Tyr Glu 180 185 190 Phe Ser Ile Asp Cys Asp Lys Ile Asn Leu PheArg Gly Val Phe Ala 195 200 205 Phe Leu Leu Tyr Val Ala Thr Phe Met TyrVal Phe Ser Thr Phe Ala 210 215 220 Asn Ile Leu Arg Asn Lys Glu Ser 225230 704 base pairs nucleic acid single linear DNA (genomic) CDS 1..699104 GTG AAA CAA AGC ACT ATT GCA CTG GCA CTC TTA CCG TTA CTG TTT ACC 48Val Lys Gln Ser Thr Ile Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr 235 240245 CCG GTG ACC AAA GCC GAG GTG TGC TCT GAA CAA GCT GAG ACC GGT CCG 96Pro Val Thr Lys Ala Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro 250 255260 TGC CGT GCA GCT ATC TTC CGC TGG TAC TTT GAC GTC ACT GAA GGT AAG 144Cys Arg Ala Ala Ile Phe Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys 265 270275 280 TGC GCT CCA TTC TTT TAC GGC GGT TGC GGC GGC AAC CGT AAC AAC TTT192 Cys Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe 285290 295 GAC ACT GAA GAG TAC TGC ATG GCA GTG TGC GGA TCC GGT GGT GGC TCT240 Asp Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Gly Gly Gly Ser 300305 310 GGT TCC GGT GAT TTT GAT TAT GAA AAG ATG GCA AAC GCT AAT AAG GGG288 Gly Ser Gly Asp Phe Asp Tyr Glu Lys Met Ala Asn Ala Asn Lys Gly 315320 325 GCT ATG ACC GAA AAT GCC GAT GAA AAC GCG CTA CAG TCT GAC GCT AAA336 Ala Met Thr Glu Asn Ala Asp Glu Asn Ala Leu Gln Ser Asp Ala Lys 330335 340 GGC AAA CTT GAT TCT GTC GCT ACT GAT TAC GGT GCT GCT ATC GAT GGT384 Gly Lys Leu Asp Ser Val Ala Thr Asp Tyr Gly Ala Ala Ile Asp Gly 345350 355 360 TTC ATT GGT GAC GTT TCC GGC CTT GCT AAT GGT AAT GGT GCT ACTGGT 432 Phe Ile Gly Asp Val Ser Gly Leu Ala Asn Gly Asn Gly Ala Thr Gly365 370 375 GAT TTT GCT GGC TCT AAT TCC CAA ATG GCT CAA GTC GGT GAC GGTGAT 480 Asp Phe Ala Gly Ser Asn Ser Gln Met Ala Gln Val Gly Asp Gly Asp380 385 390 AAT TCA CCT TTA ATG AAT AAT TTC CGT CAA TAT TTA CCT TCC CTCCCT 528 Asn Ser Pro Leu Met Asn Asn Phe Arg Gln Tyr Leu Pro Ser Leu Pro395 400 405 CAA TCG GTT GAA TGT CGC CCT TTT GTC TTT GGC GCT GGT AAA CCATAC 576 Gln Ser Val Glu Cys Arg Pro Phe Val Phe Gly Ala Gly Lys Pro Tyr410 415 420 GAA TTT TCT ATT GAT TGT GAC AAA ATA AAC TTA TTC CGT GGT GTCTTT 624 Glu Phe Ser Ile Asp Cys Asp Lys Ile Asn Leu Phe Arg Gly Val Phe425 430 435 440 GCG TTT CTT TTA TAT GTT GCC ACC TTT ATG TAT GTA TTT TCTACG TTT 672 Ala Phe Leu Leu Tyr Val Ala Thr Phe Met Tyr Val Phe Ser ThrPhe 445 450 455 GCT AAC ATA CTG CGT AAT AAG GAG TCT TAATA 704 Ala AsnIle Leu Arg Asn Lys Glu Ser 460 465 233 amino acids amino acid linearprotein 105 Val Lys Gln Ser Thr Ile Ala Leu Ala Leu Leu Pro Leu Leu PheThr 1 5 10 15 Pro Val Thr Lys Ala Glu Val Cys Ser Glu Gln Ala Glu ThrGly Pro 20 25 30 Cys Arg Ala Ala Ile Phe Arg Trp Tyr Phe Asp Val Thr GluGly Lys 35 40 45 Cys Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg AsnAsn Phe 50 55 60 Asp Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Gly GlyGly Ser 65 70 75 80 Gly Ser Gly Asp Phe Asp Tyr Glu Lys Met Ala Asn AlaAsn Lys Gly 85 90 95 Ala Met Thr Glu Asn Ala Asp Glu Asn Ala Leu Gln SerAsp Ala Lys 100 105 110 Gly Lys Leu Asp Ser Val Ala Thr Asp Tyr Gly AlaAla Ile Asp Gly 115 120 125 Phe Ile Gly Asp Val Ser Gly Leu Ala Asn GlyAsn Gly Ala Thr Gly 130 135 140 Asp Phe Ala Gly Ser Asn Ser Gln Met AlaGln Val Gly Asp Gly Asp 145 150 155 160 Asn Ser Pro Leu Met Asn Asn PheArg Gln Tyr Leu Pro Ser Leu Pro 165 170 175 Gln Ser Val Glu Cys Arg ProPhe Val Phe Gly Ala Gly Lys Pro Tyr 180 185 190 Glu Phe Ser Ile Asp CysAsp Lys Ile Asn Leu Phe Arg Gly Val Phe 195 200 205 Ala Phe Leu Leu TyrVal Ala Thr Phe Met Tyr Val Phe Ser Thr Phe 210 215 220 Ala Asn Ile LeuArg Asn Lys Glu Ser 225 230 445 base pairs nucleic acid single linearDNA (genomic) CDS 1..438 106 ATG AGA TTT CCT TCA ATT TTT ACT GCA GTT TTATTC GCA GCA TCC TCC 48 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu PheAla Ala Ser Ser 235 24 0 245 GCA TTA GCT GCT CCA GTC AAC ACT ACA ACA GAAGAT GAA ACG GCA CAA 96 Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu AspGlu Thr Ala Gln 250 255 260 265 ATT CCG GCT GAA GCT GTC ATC GGT TAC TTAGAT TTA GAA GGG GAT TTC 144 Ile Pro Ala Glu Ala Val Ile Gly Tyr Leu AspLeu Glu Gly Asp Phe 270 275 280 GAT GTT GCT GTT TTG CCA TTT TCC AAC AGCACA AAT AAC GGG TTA TTG 192 Asp Val Ala Val Leu Pro Phe Ser Asn Ser ThrAsn Asn Gly Leu Leu 285 290 295 TTT ATA AAT ACT ACT ATT GCC AGC ATT GCTGCT AAA GAA GAA GGG GTA 240 Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala AlaLys Glu Glu Gly Val 300 305 310 TCT CTA GAT AAA AGA GAG GTT GTT AGA GAGGTG TGC TCT GAA CAA GCT 288 Ser Leu Asp Lys Arg Glu Val Val Arg Glu ValCys Ser Glu Gln Ala 315 320 325 GAG ACC GGT CCG TGC CGT GCA GCT ATC TTCCGC TGG TAC TTT GAC GTC 336 Glu Thr Gly Pro Cys Arg Ala Ala Ile Phe ArgTrp Tyr Phe Asp Val 330 335 340 345 ACT GAA GGT AAG TGC GCT CCA TTC TTTTAC GGC GGT TGC GGC GGC AAC 384 Thr Glu Gly Lys Cys Ala Pro Phe Phe TyrGly Gly Cys Gly Gly Asn 350 355 360 CGT AAC AAC TTT GAC ACT GAA GAG TACTGC ATG GCA GTG TGC GGA TCC 432 Arg Asn Asn Phe Asp Thr Glu Glu Tyr CysMet Ala Val Cys Gly Ser 365 370 375 GCT ATT TAAGCTT 445 Ala Ile 146amino acids amino acid linear protein 107 Met Arg Phe Pro Ser Ile PheThr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala Ala Pro ValAsn Thr Thr Thr Glu Asp Glu Thr Ala Gln 20 25 30 Ile Pro Ala Glu Ala ValIle Gly Tyr Leu Asp Leu Glu Gly Asp Phe 35 40 45 Asp Val Ala Val Leu ProPhe Ser Asn Ser Thr Asn Asn Gly Leu Leu 50 55 60 Phe Ile Asn Thr Thr IleAla Ser Ile Ala Ala Lys Glu Glu Gly Val 65 70 75 80 Ser Leu Asp Lys ArgGlu Val Val Arg Glu Val Cys Ser Glu Gln Ala 85 90 95 Glu Thr Gly Pro CysArg Ala Ala Ile Phe Arg Trp Tyr Phe Asp Val 100 105 110 Thr Glu Gly LysCys Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn 115 120 125 Arg Asn AsnPhe Asp Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser 130 135 140 Ala Ile145 58 amino acids amino acid single linear protein 108 Arg Pro Asp PheCys Leu Glu Pro Pro Tyr Thr Gly Pro Cys Lys Ala 1 5 10 15 Arg Ile IleArg Tyr Phe Tyr Asn Ala Lys Ala Gly Leu Cys Gln Thr 20 25 30 Phe Val TyrGly Gly Cys Arg Ala Lys Arg Asn Asn Phe Lys Ser Ala 35 40 45 Glu Asp CysMet Arg Thr Cys Gly Gly Ala 50 55 56 amino acids amino acid singlelinear protein 109 Asp Phe Cys Leu Glu Pro Pro Tyr Thr Gly Pro Cys ArgAla Arg Ile 1 5 10 15 Ile Arg Tyr Phe Tyr Asn Ala Lys Ala Gly Leu CysGln Thr Phe Val 20 25 30 Tyr Gly Gly Cys Arg Ala Lys Ser Asn Asn Phe LysSer Ala Glu Asp 35 40 45 Cys Met Arg Thr Cys Gly Gly Ala 50 55 61 aminoacids amino acid single linear protein 110 Glu Val Val Arg Glu Val CysSer Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Met Ile Ser ArgTrp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr GlyGly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys MetAla Val Cys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid singlelinear protein 111 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu ProGly Pro Cys 1 5 10 15 Arg Ala Met Ile Ser Arg Trp Tyr Phe Asp Val ThrGlu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn ArgAsn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser AlaIle 50 55 60 61 amino acids amino acid single linear protein 112 Glu ValVal Arg Glu Val Cys Ser Glu Gln Ala Glu Val Gly Pro Cys 1 5 10 15 ArgAla Met Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 AlaPro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 ThrGlu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 113 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Ser Gly Pro Cys 1 5 10 15 Arg Ala Met Ile Ser Arg Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 57 amino acids amino acid single linearprotein 114 Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys Lys Ala MetIle 1 5 10 15 Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala ProPhe Phe 20 25 30 Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr GluGlu Tyr 35 40 45 Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 61 aminoacids amino acid single linear protein 115 Glu Val Val Arg Glu Val CysSer Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Gly Met Ile Ser ArgTrp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr GlyGly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys MetAla Val Cys Gly Ser Ala Ile 50 55 60 57 amino acids amino acid singlelinear protein 116 Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys ArgAla Arg Ile 1 5 10 15 Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys CysAla Pro Phe Phe 20 25 30 Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe AspThr Glu Glu Tyr 35 40 45 Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 61amino acids amino acid single linear protein 117 Glu Val Val Arg Glu ValCys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Ala Ile SerArg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe TyrGly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr CysMet Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acids amino acidsingle linear protein 118 Glu Val Val Arg Glu Val Cys Ser Glu Gln AlaGlu Thr Gly Pro Cys 1 5 10 15 Arg Ala Ile Ile Ser Arg Trp Tyr Phe AspVal Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly GlyAsn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys GlySer Ala Ile 50 55 60 61 amino acids amino acid single linear protein 119Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 1015 Arg Ala Leu Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 2530 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 4045 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 aminoacids amino acid single linear protein 120 Glu Val Val Arg Glu Val CysSer Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Ser Ile Ser ArgTrp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr GlyGly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys MetAla Val Cys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid singlelinear protein 121 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu ThrGly Pro Cys 1 5 10 15 Arg Ala Val Ile Ser Arg Trp Tyr Phe Asp Val ThrGlu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn ArgAsn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser AlaIle 50 55 60 61 amino acids amino acid single linear protein 122 Glu ValVal Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 ArgAla Gly Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 AlaPro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 ThrGlu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 123 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Met His Ser Arg Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 124 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Met Ala Ser Arg Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 125 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg AlaMet Phe Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 126 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Met Lys Ser Arg Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 127 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Met Leu Ser Arg Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 57 amino acids amino acid single linear protein 128 Glu Val CysSer Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile 1 5 10 15 Ile ArgTrp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe 20 25 30 Tyr GlyGly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr 35 40 45 Cys MetAla Val Cys Gly Ser Ala Ile 50 55 61 amino acids amino acid singlelinear protein 129 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu ThrGly Pro Cys 1 5 10 15 Arg Ala Met Ile Pro Arg Trp Tyr Phe Asp Val ThrGlu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn ArgAsn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser AlaIle 50 55 60 61 amino acids amino acid single linear protein 130 Glu ValVal Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 ArgAla Met Ile Phe Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 AlaPro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 ThrGlu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 131 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Met Ile Tyr Arg Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 132 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Met Ile Trp Arg Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 133 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg AlaMet Ile Leu Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 134 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Met Ile His Arg Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 135 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Met Ile Glu Arg Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 136 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg AlaMet Ile Gln Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 137 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Met Ile Ser Ala Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 138 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Met Ile Ser Thr Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 139 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg AlaMet Ile Ser His Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 140 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Met Ile Ser Lys Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 141 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Met Ile Ser Leu Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 142 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg AlaMet Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Val Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 143 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Met Ile Ser Arg Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Leu Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 144 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Met Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Gly Tyr Gly Gly Cys Gly Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 145 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg AlaMet Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Ala Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 146 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Met Ile Ser Arg Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysLys Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 147 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Met Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Leu Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 148 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg AlaMet Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Met Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 149 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Met Ile Ser Arg Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysAsn Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 150 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Met Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Pro Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 151 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg AlaMet Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Gln Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 57 amino acidsamino acid single linear protein 152 Glu Val Cys Ser Glu Gln Ala Glu ThrGly Pro Cys Arg Ala Met Ile 1 5 10 15 Ser Arg Trp Tyr Phe Asp Val ThrGlu Gly Lys Cys Ala Pro Phe Phe 20 25 30 Tyr Gly Gly Cys Arg Gly Asn ArgAsn Asn Phe Asp Thr Glu Glu Tyr 35 40 45 Cys Met Ala Val Cys Gly Ser AlaIle 50 55 61 amino acids amino acid single linear protein 153 Glu ValVal Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 ArgAla Met Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 AlaPro Phe Phe Tyr Gly Gly Cys Cys Gly Asn Arg Asn Asn Phe Asp 35 40 45 ThrGlu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 154 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Met Ile Ser Arg Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysSer Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 155 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Met Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Thr Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 156 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg AlaMet Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Val Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 157 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Met Ile Ser Arg Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysTyr Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 158 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Met Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Asp Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 159 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg AlaMet Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Glu Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 160 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Met Ile Ser Arg Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysHis Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 161 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Met Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Ile Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 57 amino acids amino acid single linear protein 162 Glu Val CysSer Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile 1 5 10 15 Ser ArgTrp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe 20 25 30 Tyr GlyGly Cys Gly Ala Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr 35 40 45 Cys MetAla Val Cys Gly Ser Ala Ile 50 55 61 amino acids amino acid singlelinear protein 163 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu ThrGly Pro Cys 1 5 10 15 Arg Ala Met Ile Ser Arg Trp Tyr Phe Asp Val ThrGlu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Arg Asn ArgAsn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser AlaIle 50 55 60 61 amino acids amino acid single linear protein 164 Glu ValVal Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 ArgAla Met Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 AlaPro Phe Phe Tyr Gly Gly Cys Gly Gly Ala Arg Asn Asn Phe Asp 35 40 45 ThrGlu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 57 amino acidsamino acid single linear protein 165 Glu Val Cys Ser Glu Gln Ala Glu ThrGly Pro Cys Arg Ala Met Ile 1 5 10 15 Ser Arg Trp Tyr Phe Asp Val ThrGlu Gly Lys Cys Ala Pro Phe Phe 20 25 30 Tyr Gly Gly Cys Gly Gly Asn SerAsn Asn Phe Asp Thr Glu Glu Tyr 35 40 45 Cys Met Ala Val Cys Gly Ser AlaIle 50 55 61 amino acids amino acid single linear protein 166 Glu ValVal Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 ArgAla Met Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 AlaPro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Ala Asn Asn Phe Asp 35 40 45 ThrGlu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 167 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Ala His Ser Arg Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 168 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Leu His Ser Arg Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 169 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg AlaLeu Leu Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 170 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Leu Phe Ser Arg Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 171 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Ala Ile Phe Arg Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 172 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg AlaAla Ile Trp Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 173 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Ala Ile Tyr Arg Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 174 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Leu Ile Tyr Arg Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 175 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg AlaLeu Ile Leu Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 176 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Leu Ile Pro Arg Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 177 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Leu Ile Phe Arg Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 178 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg AlaGly Ile Tyr Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 179 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Gly Ile Trp Arg Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 180 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Gly Ile Pro Arg Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 181 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg AlaAla Ile Ser Ala Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 182 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Leu Ile Ser Ala Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 183 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Ala Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Arg Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 184 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg AlaAla Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Tyr Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 185 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Met His Phe Arg Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 186 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Met His Tyr Arg Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 187 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg AlaMet His Trp Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 188 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Met Leu His Arg Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 189 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Met His Ser Arg Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Tyr Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 190 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg AlaMet Ile Phe Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Tyr Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 191 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Met Ile Tyr Arg Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysTyr Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 192 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Met Ile Trp Arg Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Tyr Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 193 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Pro Gly Pro Cys 1 5 10 15 Arg AlaLeu Ile Leu Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 194 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Gly Tyr Ile Thr Arg Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 195 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Leu His Asn Arg Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 196 lu Val Val ArgGlu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 5 10 15 rg Ala Ala HisPhe Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 la Pro Phe PheTyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 hr Glu Glu TyrCys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acids amino acidsingle linear protein 197 Glu Val Val Arg Glu Val Cys Ser Glu Gln AlaGlu Thr Gly Pro Cys 1 5 10 15 Arg Ala Leu His Phe Arg Trp Tyr Phe AspVal Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly GlyAsn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys GlySer Ala Ile 50 55 60 61 amino acids amino acid single linear protein 198Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 1015 Arg Ala Ala Leu Phe Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 2530 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 4045 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 aminoacids amino acid single linear protein 199 Glu Val Val Arg Glu Val CysSer Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Leu Phe Thr ArgTrp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr GlyGly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys MetAla Val Cys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid singlelinear protein 200 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu ThrGly Pro Cys 1 5 10 15 Arg Ala Leu Phe Lys Arg Trp Tyr Phe Asp Val ThrGlu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn ArgAsn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser AlaIle 50 55 60 61 amino acids amino acid single linear protein 201 Glu ValVal Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 ArgAla Phe Phe Lys Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 AlaPro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 ThrGlu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 202 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Ala Phe Ser Ala Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 203 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Leu Leu Ser Ala Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 204 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg AlaLeu Ile Trp His Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 205 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Leu Ile Phe Ala Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 206 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Leu Ile Tyr His Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 207 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg AlaAla Ile His Lys Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 208 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Ala Ile Tyr His Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 209 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Leu Ile Gln His Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 210 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg AlaLeu Ile Tyr Lys Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 211 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Ala Ile Gln His Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 212 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Ala Ile Phe Arg Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Arg Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 213 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg AlaAla Ile Phe Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Tyr Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 214 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Leu Ile Pro Arg Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysArg Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 215 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Val Gly ProCys 1 5 10 15 Arg Ala Leu Ile Tyr His Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 216 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Ser Gly Pro Cys 1 5 10 15 Arg AlaLeu Ile Tyr His Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 217 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Val Gly Pro Cys 1 5 10 15 Arg Ala Ala Ile Tyr His Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 218 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Ser Gly ProCys 1 5 10 15 Arg Ala Ala Ile Tyr His Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 219 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Ile Gly Pro Cys 1 5 10 15 Arg AlaLeu Ile Tyr His Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 220 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Gly Ala Ile Gln His Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 221 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Gly Ala Ile Arg His Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 222 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg GlySer Ile Arg His Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 223 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Gly Leu Ile Tyr His Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 224 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Gly Ala Ile Tyr His Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 225 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg AlaLeu His Asn Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Arg Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60 61 amino acidsamino acid single linear protein 226 Glu Val Val Arg Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg Ala Leu Phe Lys Arg Trp TyrPhe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly CysTyr Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala ValCys Gly Ser Ala Ile 50 55 60 61 amino acids amino acid single linearprotein 227 Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly ProCys 1 5 10 15 Arg Ala Leu Phe Lys Arg Trp Tyr Phe Asp Val Thr Glu GlyLys Cys 20 25 30 Ala Pro Phe Phe Tyr Gly Gly Cys Leu Gly Asn Arg Asn AsnPhe Asp 35 40 45 Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 5055 60 61 amino acids amino acid single linear protein 228 Glu Val ValArg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 1 5 10 15 Arg AlaLeu Phe Lys Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys 20 25 30 Ala ProPhe Phe Tyr Gly Gly Cys Met Gly Asn Arg Asn Asn Phe Asp 35 40 45 Thr GluGlu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile 50 55 60

What is claimed is:
 1. A protease inhibitor comprising the sequence:X¹—Val—Cys—Ser—Glu—Gln—Ala—Glu—X²—Gly—X³—Cys—Arg—Ala—X⁴—X⁵—X⁶—X⁷—Trp—Tyr—Phe—Asp—Val—Thr—Glu—Gly—Lys—Cys—Ala—Pro—Phe—X⁸—Tyr—Gly—Gly—Cys—X⁹—X¹⁰—X¹¹—X¹²—Asn—Asn—Phe—Asp—Thr—Glu—Glu—Tyr—Cys—Met—Ala—Val—Cys—Gly—Ser—Ala—Ile,wherein: X¹ is selected from Glu—Val—Val—Arg—Glu—, Asp, or Glu; X² isselected from Thr, Val, Ile and Ser; X³ is selected from Pro and Ala; X⁴is selected from Arg, Ala, Leu, Gly, or Met; X⁵ is selected from Ile,His, Leu, Lys, Ala, or Phe; X⁶ is selected from Ser, Ile, Pro, Phe, Tyr,Trp, Asn, Leu, His, Lys, or Glu; X⁷ is selected from Arg, His, or Ala;X⁸ is selected from Phe, Val, Leu, or Gly; X⁹ is selected from Gly, Ala,Lys, Pro, Arg, Leu, Met, or Tyr; X¹⁰ is selected from Ala, Arg, or Gly;X¹¹ is selected from Lys, Ala, or Asn; X¹² is selected from Ser, Ala, orArg; provided that: when X⁴ is Arg, X⁶ is Ile; when X⁹ is Arg, X⁴ is Alaor Leu; when X⁹ is Tyr, X⁴ is Ala or X⁵ is His; and either X⁵ is notIle; or X⁶ is not Ser; or X⁹ is not Leu, Phe, Met, Tyr, or Asn; or X¹⁰is not Gly; or X¹¹ is not Asn; or X¹² is not Arg.
 2. A proteaseinhibitor comprising the sequence:X¹—Val—Cys—Ser—Glu—Gln—Ala—Glu—Thr—Gly—Pro—Cys—Arg—Ala—X²—X³—X⁴—Arg—Trp—Tyr—Phe—Asp—Val—Thr—Glu—Gly—Lys—Cys—Ala—Pro—Phe—Phe—Tyr—Gly—Gly—Cys—X⁵—Gly—Asn—Arg—Asn—Asn—Phe—Asp—Thr—Glu—Glu—Tyr—Cys—Met—Ala—Val—Cys—Gly—Ser—Ala—Ile,wherein: X¹ is selected from Glu—Val—Val—Arg—Glu—, Asp, or Glu; X² isselected from Ala, Leu, Gly, or Met; X³ is selected from Ile, His, Leu,Lys, Ala, or Phe; X⁴ is selected from Ser, Ile, Pro, Phe, Tyr, Trp, Asn,Leu, His, Lys, or Glu; X⁵ is selected from Gly, Ala, Lys, Pro, Arg, Leu,Met, or Tyr; provided that: when X⁵ is Arg, X² is Ala or Leu; when X⁵ isTyr, X² is Ala or X³ is His; and either X³ is not Ile; or X⁴ is not Ser;or X⁵ is not Leu, Phe, Met, Tyr, or Asn.
 3. A protease inhibitorcomprising the sequence:Glu—Val—Val—Arg—Glu—Val—Cys—Ser—Glu—Gln—Ala—Glu—Thr—Gly—Pro—Cys—Arg—Ala—X¹—X²—X³—Arg—Trp—Tyr—Phe—Asp—Val—Thr—Glu—Gly—Lys—Cys—Ala—Pro—Phe—Phe—Tyr—Gly—Gly—Cys—X⁴—Gly—Asn—Arg—Asn—Asn—Phe—Asp—Thr—Glu—Glu—Tyr—Cys—Met—Ala—Val—Cys—Gly—Ser—Ala—Ile,wherein: X¹ is selected from Ala, Leu, Gly, or Met; X² is selected fromIle, His, Leu, Lys, Ala, or Phe; X³ is selected from Ser, Ile, Pro, Phe,Tyr, Trp, Asn, Leu, His, Lys, or Glu; X⁴ is selected from Gly, Arg, Leu,Met, or Tyr; provided that: when X¹ is Ala, X² is Ile, His, or Leu; whenX¹ is Leu, X² is Ile or His; when X¹ is Leu and X² is Ile, X³ is notSer; when X¹ is Gly, X² is Ile; when X⁴ is Arg, X¹ is Ala or Leu; whenX⁴ is Tyr, X¹ is Ala or X² is His; and either X¹ is not Met, or X² isnot Ile, or X³ is not Ser, or X⁴ is not Gly.
 4. A protease inhibitoraccording to claim 1, wherein at least two amino acid residues selectedfrom the group consisting of X⁴, X⁵, X⁶, and X⁷ differ from the residuesfound in the naturally occurring sequence of KPI.
 5. A proteaseinhibitor according to claim 1, wherein X¹ is Asp or Glu, X² is Thr, X³is Pro, and X¹² is Ser.
 6. A protease inhibitor according to claim 5,wherein X¹ is Glu, X² is Thr, X³ is Pro, X⁴ is Met, X⁵ is Ile, X⁶ isSer, X⁷ is Arg, x⁸ is Phe, X⁹ is Gly, X¹⁰ is Gly, and X¹¹ is Asn.
 7. Aprotease inhibitor according to claim 5, wherein X¹ is Asp, X² is Thr,X³ is Pro, X⁴ is Arg, X⁵ is Ile, X⁶ is Ile, X⁷ is Arg, x⁸ is Val, X⁹ isArg, X¹⁰ is Ala, and X¹¹ is Lys.
 8. A protease inhibitor according toclaim 1, wherein X¹ is Glu—Val—Val—Arg—Glu—, X² is Thr, X³ is Pro, X⁴ isMet, X⁵ is Ile, X⁶ is Ser, X⁷ is Arg, x⁸ is Phe, X⁹ is Gly, X¹⁰ is Gly,X¹¹ is Asn, and X¹² is Ala.
 9. A protease inhibitor according to claim1, wherein X¹ is Glu—Val—Val—Arg—Glu—, X² is Thr, X³ is Pro, X⁴ is Met,X⁵ is Ile, X⁶ is Ser, X⁷ is Arg, x⁸ is Phe, X⁹ is Gly, X¹⁰ is Gly, X¹¹is Ala, and X¹² is Arg.
 10. A protease inhibitor according to claim 1,wherein X¹ is Glu, X² is Thr, X³ is Pro, X⁴ is Met, X⁵ is Ile, X⁶ isSer, X⁷ is Arg, x⁸ is Phe, X⁹ is Gly, X¹⁰ is Ala, X¹¹ is Asn, and X¹² isArg.
 11. A protease inhibitor according to claim 1, wherein X¹ isGlu—Val—Val—Arg—Glu—, X² is Thr, X³ is Pro, X⁴ is Met, X⁵ is Ile, X⁶ isSer, X⁷ is Arg, x⁸ is Phe, X⁹ is Gly, X¹⁰ is Arg, X¹¹ is Asn, and X¹² isArg.
 12. A protease inhibitor according to claim 1, wherein X¹ isGlu—Val—Val—Arg—Glu—, X² is Thr, X³ is Pro, X⁴ is Met, X⁵ is Ile, X⁶ isSer, X⁷ is Arg, x⁸ is Val, Leu, or Gly, X⁹ is Gly, X¹⁰ is Gly, X¹¹ isAsn, and X¹² is Arg.
 13. A protease inhibitor according to claim 1,wherein X¹ is Glu—Val—Val—Arg—Glu—, X² is Thr, X³ is Pro, X⁴ is Met, X⁵is Ile, X⁶ is Ser, X⁷ is Ala, x⁸ is Phe, X⁹ is Gly, X¹⁰ is Gly, X¹¹ isAsn, and X¹² is Arg.
 14. A protease inhibitor according to claim 1,wherein X¹ is Glu—Val—Val—Arg—Glu—, X² is Thr, Val, or Ser, X³ is Pro,X⁴ is Ala or Leu, X⁵ is Ile, X⁶ is Tyr, X⁷ His, X⁸ is Phe, X⁹ is Gly,X¹⁰ is Gly, X¹¹ is Ala, and X¹² is Arg.
 15. A protease inhibitoraccording to claim 14, wherein X² is Thr, and X⁴ is Ala.
 16. A proteaseinhibitor according to claim 14, wherein X² is Thr, and X⁴ is Leu.
 17. Aprotease inhibitor according to claim 14, wherein X² is Val, and X⁴ isAla.
 18. A protease inhibitor according to claim 14, wherein X² is Ser,and X⁴ is Ala.
 19. A protease inhibitor according to claim 14, whereinX² is Val, and X⁴ is Leu.
 20. A protease inhibitor according to claim14, wherein X² is Ser, and X⁴ is Leu.
 21. A protease inhibitor accordingto claim 1, wherein X¹ is Glu—Val—Val—Arg—Glu—, X² is Thr, X³ is Pro, X⁴is Leu, X⁵ is Phe, X⁶ is Lys, X⁷ is Arg, X⁸ is Phe, X⁹ is Gly, X¹⁰ isGly, X¹¹ is Ala, and X¹² is Arg.
 22. A protease inhibitor according toclaim 1, wherein X¹ is Glu—Val—Val—Arg—Glu—, X² is Thr, X³ is Pro, X⁴ isLeu, X⁵ is Phe, X⁶ is Lys, X⁷ is Arg, X⁸ is Phe, X⁹ is Tyr, X¹⁰ is Gly,X¹¹ is Ala, and X¹² is Arg.
 23. A protease inhibitor according to claim1, wherein X¹ is Glu—Val—Val—Arg—Glu—, X² is Thr, X³ is Pro, X⁴ is Leu,X⁵ is Phe, X⁶ is Lys, X⁷ is Arg, X⁸ is Phe, X⁹ is Leu, X¹⁰ is Gly, X¹¹is Ala, and X¹² is Arg.
 24. A protease inhibitor according to claim 2,wherein X¹ is Glu, X² is Met, X³ is Ile, X⁴ is Ile, and X⁵ is Gly.
 25. Aprotease inhibitor according to claim 3, wherein X¹ is Met, X³ is Ser,and X⁴ is Gly.
 26. A protease inhibitor according to claim 25, whereinX² is selected from His, Ala, Phe, Lys, and Leu.
 27. A proteaseinhibitor according to claim 26, wherein X² is His.
 28. A proteaseinhibitor according to claim 27, wherein X² is Ala.
 29. A proteaseinhibitor according to claim 27, wherein X² is Phe.
 30. A proteaseinhibitor according to claim 27, wherein X² is Lys.
 31. A proteaseinhibitor according to claim 27, wherein X² is Leu.
 32. A proteaseinhibitor according to claim 3, wherein X¹ is Met, X² is Ile, and X⁴ isGly.
 33. A protease inhibitor according to claim 32, wherein X³ is Ile.34. A protease inhibitor according to claim 32, wherein X³ is Pro.
 35. Aprotease inhibitor according to claim 32, wherein X³ is Phe.
 36. Aprotease inhibitor according to claim 32, wherein X³ is Tyr.
 37. Aprotease inhibitor according to claim 32, wherein X³ is Trp.
 38. Aprotease inhibitor according to claim 32, wherein X³ is Asn.
 39. Aprotease inhibitor according to claim 32, wherein X³ is Leu.
 40. Aprotease inhibitor according to claim 32, wherein X³ is Lys.
 41. Aprotease inhibitor according to claim 32, wherein X³ is His.
 42. Aprotease inhibitor according to claim 32, wherein X³ is Glu.
 43. Aprotease inhibitor according to claim 3, wherein X¹ is Ala.
 44. Aprotease inhibitor according to claim 43, wherein X² is Ile.
 45. Aprotease inhibitor according to claim 44, wherein X³ is Phe, and X⁴ isGly.
 46. A protease inhibitor according to claim 44, wherein X³ is Tyr,and X⁴ is Gly.
 47. A protease inhibitor according to claim 44, whereinX³ is Trp, and X⁴ is Gly.
 48. A protease inhibitor according to claim44, wherein X³ is Ser or Phe, and X⁴ is Arg or Tyr.
 49. A proteaseinhibitor according to claim 43, wherein X² is His or Leu, X³ is Phe,and X⁴ is Gly.
 50. A protease inhibitor according to claim 3, wherein X¹is Leu.
 51. A protease inhibitor according to claim 50, wherein X² isHis, X³ is Asn or Phe, and X⁴ is Gly.
 52. A protease inhibitor accordingto claim 50, wherein X² is Ile, X³ is Pro, and X⁴ is Gly.
 53. A proteaseinhibitor according to claim 3, wherein X¹ is Gly, X² is Ile, X³ is Tyr,and X⁴ is Gly.
 54. A protease inhibitor according to claim 3, wherein X¹is Met, X² is His, X³ is Ser, and X⁴ is Tyr.
 55. An isolated DNAmolecule comprising a DNA sequence encoding a protease inhibitoraccording to claim
 1. 56. An isolated DNA molecule according to claim55, operably linked to a regulatory sequence that controls expression ofthe coding sequence in a host cell.
 57. An isolated DNA moleculeaccording to claim 56, further comprising a DNA sequence encoding asecretory signal peptide.
 58. An isolated DNA molecule according toclaim 57, wherein said secretory signal peptide comprises the signalsequence of yeast alpha-mating factor.
 59. A host cell transformed witha DNA molecule according to claim
 55. 60. A host cell according to claim59, wherein said host cell is E. coli or a yeast cell.
 61. A host cellaccording to claim 60, wherein said host cell is Saccharomycescerevisiae.
 62. A method for producing a protease inhibitor, comprisingthe steps of culturing a host cell according to claim 59 and isolatingand purifying said protease inhibitor.
 63. A pharmaceutical composition,comprising a protease inhibitor according to claim 1, together with apharmaceutically acceptable sterile vehicle.
 64. A method of treatmentof a clinical condition associated with increased activity of one ormore serine proteases, comprising administering to a patient sufferingfrom said clinical condition an effective amount of a pharmaceuticalcomposition according to claim
 63. 65. The method of treatment of claim64, wherein said clinical condition is blood loss during surgery.
 66. Amethod for inhibiting the activity of serine proteases of interest in amammal comprising administering a therapeutically effective dose of apharmaceutical composition according to claim
 63. 67. The method ofclaim 66, wherein said serine proteases are selected from the groupconsisting of: kallikrein; chymotrypsins A and B; trypsin; elastase;subtilisin; coagulants and procoagulants, particularly those in activeform, including coagulation factors such as factors VIIa, IXa, Xa, XIa,and XIIa; plasmin; thrombin; proteinase-3; enterokinase; acrosin;cathepsin; urokinase; and tissue plasminogen activator.
 68. A proteaseinhibitor comprising the sequence:X¹—Val—Cys—Ser—Glu—Gln—Ala—Glu—X²—Gly—Pro—Cys—Arg—Ala—X³—X⁴—X⁵—X⁶—Arg—Trp—Tyr—Phe—Asp—Val—Thr—Glu—Gly—Lys—Cys—Ala—Pro—Phe—Phe—Tyr—Gly—Gly—Cys—X⁷—Gly—Asn—Arg—Asn—Asn—Phe—Asp—Thr—Glu—Glu—Tyr—Cys—Met—Ala—Val—Cys—Gly—Ser—Ala—Ile,wherein: X¹ is selected from Glu—Val—Val—Arg—Glu—, Asp, or Glu; X² isselected from Thr, Val, Ile and Ser; X³ is selected from Arg, Ala, Leu,Gly, or Met; X⁴ is selected from Ile, His, Leu, Lys, Ala, or Phe; X⁵ isselected from Ser, Ile, Pro, Phe, Tyr, Trp, Asn, Leu, His, Lys, or Glu;X⁶ is selected from Arg, His, or Ala; and X⁷ is selected from Gly, Ala,Lys, Pro, Arg, Leu, Met, or Tyr.
 69. A protease inhibitor according toclaim 68, wherein at least two amino acid residues selected from thegroup consisting of X³, X⁴, X⁵, and X⁶ differ from the residues found inthe naturally occurring sequence of KPI.
 70. A protease inhibitoraccording to claim 68, wherein X¹ is Glu—Val—Val—Arg—Glu—, X² is Thr,Val, or Ser, X³ is Ala or Leu, X⁴ is Ile, X⁵ is Tyr, X⁶ is His and X⁷isGly.
 71. A protease inhibitor according to claim 70, wherein X² is Thr,and X³ is Ala.
 72. A protease inhibitor according to claim 70, whereinX² is Thr, and X³ is Leu.
 73. A protease inhibitor according to claim70, wherein X² is Val, and X³ is Ala.
 74. A protease inhibitor accordingto claim 70, wherein X² is Ser, and X³ is Ala.
 75. A protease inhibitoraccording to claim 70, wherein X² is Val, and X³ is Leu.
 76. A proteaseinhibitor according to claim 70, wherein X² is Ser, and X³ is Leu.
 77. Aprotease inhibitor according to claim 68, wherein X¹ isGlu—Val—Val—Arg—Glu—, X² is Thr, X³ is Leu, X⁴ is Phe, X⁵ is Lys, X⁶ isArg and X⁷ is Gly.
 78. A protease inhibitor according to claim 68,wherein X¹ is Glu—Val—Val—Arg—Glu—, X² is Thr, X³ is Leu, X⁴ is Phe, X⁵is Lys, X⁶ is Arg and X⁷ is Tyr.
 79. A protease inhibitor according toclaim 68, wherein X¹ is Glu—Val—Val—Arg—Glu—, X² is Thr, X³ is Leu, X⁴is Phe, X⁵ is Lys, X⁶ is Arg and X⁷ is Leu.